Real-time Fluorescent Quantitative PCR Detection Research Articles

Establishment of a real-time fluorescent quantitative PCR detection method and phylogenetic analysis of BoAHV-1

BackgroundInfectious bovine rhinotracheitis (IBR), caused by Bovine alphaherpesvirus-1 (BoAHV-1), is an acute, highly contagious disease primarily characterized by respiratory tract lesions in infected cattle. Due to its severe pathological damage and extensive transmission, it results in significant economic losses in the cattle industry. Accurate detection of BoAHV-1 is of paramount importance. In this study, we developed a real-time fluorescent quantitative PCR detection method for detecting BoAHV-1 infections. Utilizing this method, we tested clinical samples and successfully identified and isolated a strain of BoAHV-1.1 from positive samples. Subsequently, we conducted a genetic evolution analysis on the isolate strain’s gC, TK, gG, gD, and gE genes.ResultsThe study developed a real-time quantitative PCR detection method using SYBR Green II, achieving a detection limit of 7.8 × 101 DNA copies/μL. Specificity and repeatability analyses demonstrated no cross-reactivity with other related pathogens, highlighting excellent repeatability. Using this method, 15 out of 86 clinical nasal swab samples from cattle were found to be positive (17.44%), which was higher than the results obtained from conventional PCR detection (13.95%, 12/86). The homology analysis and phylogenetic tree analysis of the gC, TK, gG, gD, and gE genes of the isolated strain indicate that the JL5 strain shares high homology with the BoAHV-1.1 reference strains. Amino acid sequence analysis revealed that gC, gE, and gG each had two amino acid mutations, while the TK gene had one synonymous mutation and one H to Y mutation, with no amino acid mutations observed in the gD gene. Phylogenetic tree analysis indicated that the JL5 strain belongs to the BoAHV-1.1 genotype and is closely related to American strains such as C33, C14, and C28.ConclusionsThe established real-time fluorescent quantitative PCR detection method exhibits good repeatability, specificity, and sensitivity. Furthermore, genetic evolution analysis of the isolated BoAHV-1 JL-5 strain indicates that it belongs to the BoAHV-1.1 subtype. These findings provide a foundation and data for the detection, prevention, and control Infectious Bovine Rhinotracheitis.

Establishment and Evaluation Strategy of an in vitro Cell Model of Bone Marrow Microenvironment Injury in Mouse Acute Graft-Versus-Host Disease

To establish a mesenchymal stem cell（MSC）-based in vitro cell model for the evaluation of mouse bone marrow acute graft-versus-host disease (aGVHD). Female C57BL/6N mice aged 6-8 weeks were used as bone marrow and lymphocyte donors, and female BALB/c mice aged 6-8 weeks were used as aGVHD recipients. The recipient mouse received a lethal dose (8.0 Gy,72.76 cGy/min) of total body γ irradiation, and injected with donor mouse derived bone marrow cells (1×107/mouse) in 6-8 hours post irradiation to establish a bone marrow transplantation (BMT) mouse model (n=20). In addition, the recipient mice received a lethal dose (8.0 Gy,72.76 cGy/min) of total body γ irradiation, and injected with donor mouse derived bone marrow cells (1×107/mouse) and spleen lymphocytes (2×106/mouse) in 6-8 hours post irradiation to establish a mouse aGVHD model (n=20). On the day 7 after modeling, the recipient mice were anesthetized and the blood was harvested post eyeball enucleation. The serum was collected by centrifugation. Mouse MSCs were isolated and cultured with the addition of 2%, 5%, and 10% recipient serum from BMT group or aGVHD group respectively. The colony-forming unit-fibroblast（CFU-F) experiment was performed to evaluate the potential effects of serums on the self-renewal ability of MSC. The expression of CD29 and CD105 of MSC was evaluated by immunofluorescence staining. In addition, the expression of self-renewal-related genes including Oct-4, Sox-2, and Nanog in MSC was detected by real-time fluorescence quantitative PCR（RT-qPCR). We successfully established an in vitro cell model that could mimic the bone marrow microenvironment damage of the mouse with aGVHD. CFU-F assay showed that, on day 7 after the culture, compared with the BMT group, MSC colony formation ability of aGVHD serum concentrations groups of 2% and 5% was significantly reduced （P < 0．05）; after the culture, at day 14, compared with the BMT group, MSC colony formation ability in different aGVHD serum concentration was significantly reduced （P < 0．05）. The immunofluorescence staining showed that, compared with the BMT group, the proportion of MSC surface molecules CD29+ and CD105+ cells was significantly dereased in the aGVHD serum concentration group (P < 0.05), the most significant difference was at a serum concentration of 10% （P < 0．001, P < 0.01）. The results of RT-qPCR detection showed that the expression of the MSC self-renewal-related genes Oct-4, Sox-2, and Nanog was decreased, the most significant difference was observed at an aGVHD serum concentration of 10% （P < 0.01,P < 0．001,P < 0．001）. By co-culturing different concentrations of mouse aGVHD serum and mouse MSC, we found that the addition of mouse aGVHD serum at different concentrations impaired the MSC self-renewal ability, which providing a new tool for the field of aGVHD bone marrow microenvironment damage.

Rapid detection of grass carp reovirus type 1 using RPA-based test strips combined with CRISPR Cas13a system.

Due to the existence of grass carp reovirus (GCRV), grass carp hemorrhagic disease occurs frequently, and its high pathogenicity and infectivity are great challenges to the aquaculture industry. As a highly pathogenic pathogen, the outbreak of hemorrhagic disease often causes tremendous economic losses. Therefore, it is important to rapidly and accurately detect GCRV on site to control timely. In this study, recombinant enzyme amplification (RPA) combined with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13a system was employed to establish a method to detect the vp7 gene of grass carp reovirus type 1. This method can be adopted for judging the results by collecting fluorescence signal, ultraviolet excitation visual fluorescence and test strip. Combined with the RPA amplification experiment, the detection limit of the RPA-CRISPR method can reach 7.2 × 101 copies/μL of vp7 gene per reaction, and the detection process can be completed within 1 h. In addition, this method had no cross-reaction with the other 11 common aquatic pathogens. Then, the performance of the RPA-CRISPR/Cas13a detection method was evaluated by comparing it with the real-time fluorescence quantitative PCR detection method of clinical samples. The results of RPA-CRISPR/Cas13a detection were shown to be in consistence with the results obtained from the real-time fluorescence quantitative PCR detection. The coincidence rate of this method with 26 GCRV clinical samples was 92.31%. In summary, this method has high sensitivity, specificity and on-site practicability for detecting GCRV type 1, and has great application potential in on-site GCRV monitoring.

Low-energy LED red light inhibits the NF-κB pathway and promotes hPDLSCs proliferation and osteogenesis in a TNF-α environment in vitro.

There are few studies on the effect of low-energy LED red light on periodontal tissue regeneration in an inflammatory environment. In this study, Cell Counting Kit-8(CCK-8) assays were used to detect the effects of TNF-α at three different concentrations (0, 10ng/ml, and 20ng/ml) on the proliferation of human periodontal ligament stem cells (hPDLSCs), and 10ng/ml was selected as the subsequent experimental stimulation concentration. CCK-8 assays were used to detect the effect of LED red light with energy density of 1J/ cm2, 3J/ cm2, and 5J/cm2 on the proliferation of hPDLSCs. The promotion effect of energy density of 5J/cm2 on the proliferation of hPDLSCS was the most obvious (p < 0.05). Set CON group, ODM group, ODM + 10ng/ml TNF-α group, and ODM + 10ng/ml TNF-α + 5J/ cm2 LED red light group. Alkaline phosphatase staining and activity detection, alizarin red staining and calcium nodules quantitative detection of osteoblast differentiation products, real-time fluorescence quantitative PCR detection of osteoblast gene expression (Runx2, Col-I, OPN, OCN). The results showed that ODM showed the strongest osteoblast ability, followed by ODM + 10ng/ml TNF-α + 5J/ cm2 LED red light group. The osteoblast ability of ODM + 10ng/ml TNF-α was decreased, but was not found in CON group. Western blot was used to detect the expression of NF-κB pathway protein and osteoblast-related proteins (Runx2, Col-I, OPN, OCN) after addition of PDTC inhibitor. The results showed that the expression of p-IκBα was increased and the expression of IκBα was decreased (p < 0.05). The expression of osteoblast protein increased after the addition of inhibitor (p < 0.05). Therefore, in an inflammatory environment constructed by 10ng/ml TNF-α, 5J/cm2 LED red light can upregulate the proliferation and osteogenesis of hPDLSCs by inhibiting NF-κB signaling pathway.

Development of a Quantitative PCR Method for Specific and Quantitative Detection of Enterocytospora artemiae, a Microsporidian Parasite of Chinese Grass Shrimp (Palaemonetes sinensis)

Enterocytospora artemiae (EAM) mainly parasitizes the hepatopancreas of Palaemonetes sinensis. Serious infection leads to hepatopancreatic lesions, which greatly reduce the vitality of P. sinensis. Currently, EAM is detected via conventional PCR methods. However, conventional PCR has low sensitivity and cannot be used for accurate quantitative detection of EAM or its parasitic activity in host tissues. In this study, we designed a pair of specific primers based on the sequence of the ribosomal protein S9 gene (RPS9; GenBank accession number: MZ420734) to establish and optimize a SYBR Green I real-time fluorescent quantitative PCR detection method for EAM. Only EAM appeared as a bright and single target band, whereas other microorganisms did not, indicating that the primer for RPS9 had high specificity. This method displayed optimum amplification effects at an annealing temperature of 55°C, and the melting curve of the product produced a single peak. The established method showed a good linear relationship from 2.2 × 108 to 2.2 × 101 copies/μL. The relationship between the number of cycle thresholds (Ct) and the logarithm of the initial template amount (x) conformed to Ct = −3.281 log x + 36.543 (R2 = 0.998). Amplification efficiency was 101.737%, and the lower limit of detection sensitivity was 2.2 × 101 copies/μL. Good intra- and inter-group repeatability was observed within the linear range. The sensitivity of this method was more than 200 times higher than that of nested PCR. Thus, detection data obtained using this method may be useful as a technical reference for rapid and accurate identification of EAM infection and for the prevention and control of EAM during P. sinensis breeding.

Colonization Characteristics of Poplar Fungal Disease Biocontrol Bacteria N6-34 and the Inhibitory Effect on Pathogenic Fungi by Real-Time Fluorescence Quantitative PCR Detection.

Botryosphaeria dothidea is one of the most important diseases which can cause poplar canker. In our previous study, the endophytic Bacillus subtilis N6-34 screened from poplar tissue was found to be an antagonistic strain against B. dothidea. In order to ascertain the colonization rule of B. subtilis N6-34 in poplar plants, colonization of B. subtilis N6-34 labeled with a green fluorescent protein (GFP) was investigated in poplar plants and the rhizosphere soil. To confirm the inhibitory effect of the strain N6-34 on pathogenic fungi, real-time fluorescent quantitative PCR experiment with Fusarium oxysporum as the target strain was carried out. Firstly, a plasmid (pHT01-P43GFPmut3a) containing gfp gene was successfully transformed into wild B. subtilis N6-34, which has the similar characteristics with the strain N6-34 in cell growth and antifungal activity. The poplar pot experiments were carried out to examine the colonization rules and colonization quantity in poplar plants and rhizosphere soil. Observation with a confocal laser scanning microscope showed that GFP-labeled B. subtilis N6-34 (N6-34-GFP) could colonize in primary root, lateral root and adventitious root. With the extension of inoculation time, the colonization quantity of N6-34-GFP in the rhizosphere soil and poplar plants showed a trend of first increasing, then stabilizing for a period of time and then decreasing. The real-time fluorescent quantitative PCR result showed a gradual decrease in the number of F. oxysporum with increasing inoculation time. Therefore, N6-34-GFP exhibited colonization in the rhizosphere soil and different parts of poplar plants. In addition, the strain N6-34 could inhibit the growth of pathogenic fungi. The ability of B. subtilis N6-34 to colonize in the rhizosphere soil and poplar plants and to inhibit fungal growth in vitro suggest a potential application of this strain as a biological control agent.

Detection of microamounts of novel coronavirus residues in environment by digital PCR

<p indent="0mm">2019 novel coronaviruses (2019-nCoV) swept the world and are still at a high incidence stage. Highly sensitive and accurate nucleic acid molecular diagnostic techniques and methods have become a hot spot of global concern. 2019-nCoV is a kind of RNA virus. At present, there are many methods to detect virus RNA, including real-time fluorescence quantitative PCR (RT-PCR), colloidal gold detection, chemiluminescence detection, and so on. Colloidal gold detection and chemiluminescence detection need to use monoclonal antibodies against 2019-nCoV, but there are some problems in the preparation of 2019-nCoV monoclonal antibodies, such as long cycle, complex preparation and easy pollution. So the above two methods are not easy to be established for the detection of 2019-nCoV. The superiority of PCR technology is widely used in auxiliary medical clinical diagnosis, customs inspection and quarantine, the development of new agriculture, national defense military defense, and basic research of bioscience, and so on. However, the quantification of the target DNA fragment in RT-PCR is still relatively quantitative. Because this technique mainly depends on CT value and standard curve to quantify the target DNA in the experiment, which affects the amplification efficiency of the whole system, and the sensitivity and accuracy of the reaction system are greatly affected. Because the 2019-nCoV virus is highly contagious, the detection methodology is required to be highly sensitive, which is in line with the characteristics of digital PCR (droplet digital PCR system, referred to as ddPCR). ddPCR has the advantages of absolute quantification, high specificity, high sensitivity and strong interference ability, which can better combine molecular biology and medicine closely, and has a great advantage in the detection of 2019-nCoV. In this study, based on droplet digital PCR detection technology, the detection reagent and method were established using the principle of nested PCR. The Saliva of 333 2019-nCoV patients and their environmental samples were detected and compared with real-time PCR (RT-PCR). The linear range of the droplet digital PCR method established in this study was between 25–5 copies/µL, and minimum detection limit was 0.5 copies/µL, which was 2–3 orders of magnitude higher than that of the commercial real-time fluorescent quantitative PCR detection technology. In 333 samples, 7 were positive in 197 environmental samples, and 9 were positive in 136 human samples. The detection accuracy was 100%, and the environmental micro-virus residues that could not be detected by RT-PCR were detected. The digital PCR detection method established in this study was highly specific, and the minimum detection limit was far lower than that of RT-PCR. It is of great significance for sensitive and accurate detection of 2019-nCoV infection, early infection and environmental microvirus.

Molecular and Toxicity Analyses of White Granulated Sugar and Other Processing Products Derived From Transgenic Sugarcane.

This study aimed to prepare the sugar industry for the possible introduction of genetically modified (GM) sugarcane and derived retail sugar products and to address several potential public concerns regarding the characteristics and safety of these products. GM sugarcane lines with integrated Cry1Ab and EPSPS foreign genes were used for GM sugar production. Traditional PCR, real-time fluorescent quantitative PCR (RT-qPCR), and enzyme-linked immunosorbent assay (ELISA) were performed in analyzing leaves, stems, and other derived materials during sugar production, such as fibers, clarified juices, filter mud, syrups, molasses, and final GM sugar product. The toxicity of GM sugar was examined with a feeding bioassay using Helicoverpa armigera larvae. PCR and RT-qPCR results showed that the leaves, stems, fibers, juices, syrups, filter mud, molasses, and white granulated sugar from GM sugarcane can be distinguished from those derived from non-GM sugarcane. The RT-qPCR detection method using short amplified product primers was more accurate than the traditional PCR method. Molecular analysis results indicated that trace amounts of DNA residues remain in GM sugar, and thus it can be accurately characterized using molecular analysis methods. ELISA results showed that only the leaves, stems, fibers, and juices sampled from the GM sugarcane differed from those derived from the non-GM sugarcane, indicating that filter mud, syrup, molasses, and white sugar did not contain detectable Cry1Ab and EPSPS proteins. Toxicity analysis showed that the GM sugar was not toxic to the H. armigera larvae. The final results showed that the GM sugar had no active proteins despite containing trace amounts of DNA residues. This finding will help to pave the way for the commercialization of GM sugarcane and production of GM sugar.

LncRNA LOC101927514 regulates PM2.5-driven inflammation in human bronchial epithelial cells through binding p-STAT3 protein

Long-term exposure to fine particulate matter (PM2.5) may cause or exacerbate many diseases, including respiratory inflammation. However, the full mechanism is not yet fully understood. The newly discovered long chain non-coding RNA, though unable to encode proteins, regulates multiple life activities and participates in the development of inflammation. In this study, we set up a cell inflammation model by using normal bronchial 16HBE cells exposed to PM2.5. High-throughput sequencing, as well as real-time fluorescent quantitative PCR detection and validation, was performed on the inflamed cells to evaluate the expression level of long chain noncoding RNA that helped us to identify the LncRNA LOC101927514. Inhibiting LncRNA LOC101927514 expression by RNAi, reflected in a reduction in inflammation, is driven by PM2.5. In addition, we identify LncRNA LOC101927514 to be located within the nucleus and binds to STAT3, altering the inflammatory state of the cells and IL6 and IL8 release. This study identifies that LncRNA LOC101927514 is a new potential target for future treatment of the inflammatory response activated by PM2.5 in the respiratory system.

Maternal Blood Milk Saliva Sample Selection and the Transmission of Hepatitis B Virus Infectious Research

Background: Hepatitis B is an infectious disease, which is a main way of vertical transmission of infectious HBV between mother and infant. Hepatitis B virus infection is always a hot topic of social concern, especially in China. The paper studies hepatitis B virus in maternal blood, breast milk, saliva of hepatitis B virus infection model (HBV-M) in Hefei city, Anhui province, PRC. HBV-DNA load and related data in Hefei city are used for risk assessment of the transmission of hepatitis B virus to provide evidence for evidence-based medicine and scientific guidance of infant feeding patterns. Methods: On the principle of informed consent, inpatient hepatitis B maternal blood 695, breast milk, saliva 614,169 copies were used as the object of analysis, using the ELISA method for the detection of HBV-M, using real-time fluorescence quantitative PCR detection of HBV-DNA load. We analyze HBsAg in saliva, milk, the positive rate of HBV-DNA and HBV-M in serum, saliva, milk, and explore the positive rate of HBV-DNA and serum HBV-DNA load correlation. Results: At the age of 18 - 44 years old perinatal women, HBV-DNA positive rates of maternal serum, breast milk, saliva were 157 cases in A group HBsAg, HBeAg positive: 99.36%, 88.06%, 96.77%; in 312 cases in group B, HBeAb HBsAg, HBcAb positive: 17.63%, 2.93%, 54.67%; 69 cases in C group HBsAg, HBcAb positive: 63.77%, 27.27%, 28.57%; D group of 71 patients with simple HBcAb positive: 12.68%, 3.13%, 0%; E group and 86 cases in control group HBVM: 1.16%, 0%, 0%. According to the serum and milk testing of Group A and Group B, HBV-DNA chi-square is χ2 = 237.45, P ; there is a significant difference in serum and saliva; HBV-DNA chi-square χ2 = 289.49, P Conclusion: 1) HBV-DNA load high maternal blood, breast milk, saliva are potentially persistent hepatitis B virus infection risk, especially infectious blood. 2) Of maternal milk, saliva and blood HBV-DNA HBV-DNA load were positively correlated (r = 0.96; P ing, breast milk and saliva HBV-DNA positive rates were increased and infectivity enhanced. 3) Maternal blood, breast milk, saliva specimens for any HBV-DNA ≥ 1000 copies/ml are not breastfeeding. 4) The mother who carries the hepatitis B virus cannot do maternal infant feeding, and deep kiss intimate contact, in order to prevent blood, saliva and other ways of infection of hepatitis B virus. 5) Saliva testing is instead of milk inspection, because saliva is easier;</sp

Detection of Aspergillus With Real-Time Fluorescent Quantitative PCR

Objective: To establish a real-time fluorescent quantitative PCR (FQ-PCR) detection system for aspergillus in order to quickly, accurately detect common aspergillus pathogens in clinically invasive fungal infections.Methods: Standard strains of cultured fungi were used to prepare spore suspension samples with a concentration of 1,000,000 spores/mL. DNA samples were extracted with a one-step lysing heating method (LHM). Primers and probes for aspergillus fumigatus (A. fumigatus), aspergillus flavus (A. flavus), aspergillus niger (A. niger), and aspergillus terreus (A. terreus) were designed with the Primer Premier 5.0 and Beacon Designer 7.7. The most effective primer probe was obtained after an FQ-PCR detection system screening which tested the efficiency and amplification effect for extracting aspergillus DNA via a one-step LHM, and verified specificity, repeatability, and sensitivity of the reaction system.Results: No non-specific amplification was found. Coefficient of variation (CV) of Ct values in repetitive intra- and inter-assay experiments were less than 5 % (0.37 % - 3.53 %). All four aspergillum sensitivities reached at least 35 spores/mL after the established reaction system was detected.Conclusion: 1. Samples were prepared with spores cultured from aspergillus standard strains. Aspergillus DNA was extracted with an LHM method and was applied to the real-time fluorescent quantitative PCR detection. 2. Primers and probes for aspergillus fumigatus, aspergillus flavus, aspergillus niger, and aspergillus terreus were successfully designed and screened. Real-time FQ-PCR detection system for aspergillus was established. It was highly specific, sensitive and repeatable.

The role of glutathione in steroid induced bone marrow mesenchymal stem cells dysfunction

To investigate the protective effect of the antioxidant glutathione (GSH) on the steroid-induced imbalance between osteogenesis and adipogenesis in human bone marrow mesenchymal stem cells (BMSCs). The BMSCs were isolated from the proximal femur bone marrow from 3 patients of femoral neck fracture and were separated, cultured, and purificated by density gradient centrifugation and adherent wall method in vitro. The third generation BMSCs were divided into 5 groups: group A, BMSCs (1×10 5 cells/mL); group B, BMSCs (1×10 5 cells/mL)+10 μmol/L dexamethasone; group C, BMSCs (1×10 5 cells/mL)+10 μmol/L dexamethasone+5 μmol/L GSH; group D, BMSCs (1×10 5 cells/mL)+10 μmol/L dexamethasone+10 μmol/L GSH; group E, BMSCs (1×10 5 cells/mL)+10 μmol/L dexamethasone+50 μmol/L GSH. After cultured for 7 days, the reactive oxygen species expression was detected by flow cytometry; the superoxide dismutase (SOD) and Catalase mRNA expressions were determined by RT-PCR; the peroxisome proliferator-activated receptors γ (PPAR-γ), CCAAT/enhancer-binding family of proteins (C/EBP), Runx2, and alkaline phosphatase (ALP) mRNA expressions were evaluated by real-time fluorescence quantitative PCR. After cultured for 21 days, Oil red O staining was used to observe the adipogenesis differentiation of cells, and the expressions of related proteins were detected by Western blot. The reactive oxygen species expression in group B was obviously higher than in the other groups, in group C than in groups A, D, and E, and in groups D, E than in group A, all showing significant differences between groups ( P<0.05); but there was no significant difference between groups D and E ( P>0.05). The oil red O staining positive cells in group B were obviously more than the other groups, and groups C, D, E, and A decreased sequentially, the absorbance ( A) values had significant differences between groups ( P<0.05). RT-PCR detection showed that the relative expressions of SOD and Catalase mRNA in group B were significantly lower than those in the other groups, while in group C than in groups A, D, and E ( P<0.05), but there was no significant difference among groups A, D, and E ( P>0.05). Real-time fluorescence quantitative PCR detection showed that the relative expressions of PPAR-γ and C/EBP mRNA in group B were significantly higher than those in the other groups, while in group C than in groups A, D, and E, and in groups D, E than in group A ( P<0.05); but there was no significant difference between groups D and E ( P>0.05). The relative expressions of Runx2 and ALP mRNA in group B were significantly lower than those in the other groups, while in group C than in groups A, D, and E, and in groups D, E than in group A ( P<0.05); but there was no significant difference between groups D and E ( P>0.05). Western blot detection showed that the relative expression of PPAR-γ and C/EBP protein in group B was significantly higher than those in the other groups, and groups C, D, E, and A decreased sequentially, all showing significant differences between groups ( P<0.05). The relative expression of Runx2 and ALP protein in group B was significantly lower than those in the other groups, and groups C, D, E, and A increased sequentially, all showing significant differences between groups ( P<0.05). GSH can inhibit the adipogenesis differentiation and enhance the osteogenic differentiation of human BMSCs by reducing the intracellular reactive oxygen species level; and in a certain range, the higher the concentration of GSH, the more obvious the effect is.

Expression of Sclerostin in medial and lateral subchondral bone of the varus osteoarthritic knee plateau

To study the expression difference of Sclerostin in the medial and lateral subchondral bone of the varus osteoarthritic knee plateau. The tibial plateau was obtained from 20 patients with varus knee osteoarthritis receiving total knee arthroplasty from March to October 2015. There were 8 males and 12 females with an average age of 67.8 years (range, 61-78 years). The mean course of osteoarthritis was 3.2 years (range, 2-5 years). Before operation, the varus angle was 12.0-25.5° (mean, 17.6°) on the X-ray film. Five cases were rated as grade III and 15 cases as grade IV according to Kellgren-Lawrance classification. Micro-CT scan was performed on the medial and lateral subchondral bone to compare the changes of bone structure; bone volume/total volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), structure model index (SMI), and the trabecular separation (Tb.Sp) were measured. Immunohistochemistry and real-time fluorescent quantitative PCR were used to test the expressions of Sclerostin protein and sost gene. Micro-CT showed that BV/TV, Tb.N, and Tb.Th significantly increased in the medial subchondral bone when compared with the lateral part ( P<0.05), but SMI and Tb.Sp significantly reduced ( P<0.05). Real-time fluorescent quantitative PCR detection showed that sost gene expression level in the medial subchondral bone (1.000) was significantly lower than that in the lateral part (4.157±2.790) ( t=2.371, P=0.040). The percentage of Sclerostin positive cells in the lateral subchondral bone (52.00%±0.19%) was significantly higher than that in the medial subchondral bone (7.20%±0.04%) ( t=5.094, P=0.005). Sclerostin plays an important role in the subchondral bone remodeling of the varus osteoarthritic knee. And the low expression of Sclerostin may be an important factor to promote bone remodeling and aggravate knee deformity.

EXPERIMENTAL STUDY ON OSTEOGENIC ACTIVITY OF RABBIT BONE MARROW MESENCHYMAL STEM CELLS INDUCED BY KLD-12 POLYPEPTIDE/RECOMBINANT HUMAN BONE MORPHOGENETIC PROTEIN 2 GEL

To investigate the effect of KLD-12 polypeptide complexed with recombinant human bone morphogenetic protein 2 (rhBMP-2) on osteogenic activity of rabbit bone marrow mesechymal stem cells (BMSCs). Bone marrow was harvested from 3-month-old New Zealand white rabbit, and density gradient method was used to isolate and culture BMSCs. The third generation BMSCs were used for three-dimensional culture of KLD-12 polypetide/rhBMP-2 in vitro (experimental group) and KLD-12 polypeptide (control group). The morphology of the cells in the gel was observed by inverted phase contrast microscope at 7 days; alkaline phosphatase (ALP) and osteocalcin protein content were dectected at 3, 7, 10, 14, and 21 days; collagen type I immunofluorescence staining was done and real-time fluorescent quantitative PCR was performed to detect the relative expression of collagen type I and osteocalcin gene at 14 days. Under the inverted phase contrast microscope, the BMSCs in the gel of the experimental group and the control group showed circular growth, and the distribution was uniform at 7 days. There was no significant difference in the expressions of ALP and osteocalcin protein content between 2 groups at 3 and 7 days (P>0.05); the above indexes in experimental group were significantly higher than those in the control group at 10-21 days (P<0.05). Laser scanning confocal microscope observation showed that immunofluorescence staining for collagen type I was positive in the experimental group, and the expression was higher than that in the control group at 14 days. Real-time fluorescence quantitative PCR detection showed that the collagen type I and osteocalcin gene expressions were significantly higher than those in the control group (t=15.902, P=0.000; t=12.998, P=0.000). BMSCs can normally grow and proliferate in the KLD-12 polypeptide, and KLD-12 polypeptide/rhBMP-2 has good biological activity to induce BMSCs differentiation into osteoblasts.

Ginkgo biloba extract enhances chemotherapy sensitivity and reverses chemoresistance through suppression of the KSR1-mediated ERK1/2 pathway in gastric cancer cells.

Kinase suppressor of Ras 1 (KSR1) is a scaffold protein that modulates the activation of the oncogenic mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling pathway. Ginkgo biloba extract (EGb) 761 has been demonstrated to possess antitumor activity that may be related to the KSR1-mediated ERK signaling pathway. However, the roles and its underlying mechanism in gastric cancer are unclear. In the present study, 62 gastric cancer and matched normal tissues were exploited for immunohistochemistry and real-time fluorescent quantitative PCR detection. Results of the immunohistochemistry showed that the expression of ERK1/2 and p-ERK1/2 was correlated to the expression of KSR1 and p-KSR1 in the gastric cancer tissues, and the overexpression of KSR1, p-KSR1, ERK1/2 and p-ERK1/2 was significantly associated with histological grade, TNM stage, lymph node and distant metastasis. Compared with the normal tissues, the relative mRNA copy values of KSR1, ERK1 and ERK2 in the cancer tissues were 2.43 ± 0.49, 2.10 ± 0.44 and 3.65 ± 0.94. In addition, the expression of KSR1, p-KSR1, ERK1/2 and p-ERK1/2 in human gastric cancer multidrug resistant SGC-7901/CDDP cells was higher than that in the SGC-7901 cells as detected by the methods of immunocytochemistry and western blot analysis. EGb 761 not only suppressed expression of these proteins induced by cisplatin (CDDP) and etoposide in SGC-7901 cells, but also inhibited expression of these proteins in the SGC-7901/CDDP cells. Meanwhile, the proliferation-suppressing and apoptosis-inducing capacities of CDDP and etoposide were enhanced following combined treatment with EGb 761. Moreover, EGb 761 reduced the malondialdehyde (MDA) content and elevated the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in the tumor cells. These results confirmed that activation of the KSR1-mediated ERK1/2 signaling pathway may contribute to tumorigenesis, metastasis and chemoresistance of human gastric cancer. EGb 761 enhanced the chemotherapy sensitivity and reversed the chemoresistance through suppression of the KSR1-mediated ERK1/2 pathway in gastric cancer cells, and the underlying mechanism may be related to its antioxidative activity.

Clinical Significance of the detection of HBV-DNA by the Real-time Fluorescence quantitative PCR

Objective To analyze HBV-DNA results of hepatitis B patients using real-time fluorescence quantitative PCR detection and to explore the significance of their clinical treatment. Methods 850 cases of clinical specimens were detected by time-resolved fluorescence immunoassay quantitative determination and fluorescent quantitative PCR assay detected HBV-DNA of blood samples and the results were divided groups. Results There were 261 positive in group that the HBsAg( + ), HBeAg ( + )and HBcAb ( + )were positive and positive rate was 86.4% (261/302)and its quantitative PCR for copy number was(2. 21 ± 0. 76) × 107/ml.There were 96 positive in group that the HBsAg( + ) ,HBeAb( + )and HBcAb( + )were positive and the positive rate was 29.91% (96/321)and its quantitative PCR for copy number was( 1.69 ± 0. 98) × 106/ml. There were 9 were positive in group that the HBsAg( + )and HBcAb( + )were positive and positive rate was 28. 13% (9/32). There were 4 positive in group that the HBeAb ( + ) and HBcAb ( + ) were positive and positive rate was 14. 29% (4/28). There were 6 positive in group that the HBcAb( + )was positive and the positive rate was 42.9% (6/11). There were 5 positive in group that the HBsAb( + ), HBeAb( + ) and HBcAb( + ) were positive and the positive rate was 6. 75% (5/74). There were 2 positive in group that HBsAb( + )and HBeAb( + )positive and the positive rate was 14. 3% (2/13). There were 1 positive in group that HBsAb( + ) and HBcAb ( + )were positive and a positive rate was 7.69% (1/12). 4 were negative in group of HBsAb( + ). There were 21 positive in the group that were HBsAg( + ), HBsAb( + ), HBeAg( + )and HBcAb( + )were positive and the positive rate was 87.50% (21/24). The positive rate was 100% 1 in group of HBsAg( + ). There were 3 positive in group that HBsAg( + ), HBsAb ( + ), HBeAb ( + ) and HBcAb ( + ) were positive and the positive rate was 30. 00% (3/10). The remaining 38 patients were negative results. Conclusion PCR quantitative determination of HBV-DNA can be a true reflection of hepatitis B virus infection and replication in vivo and more conducive to clinical treatment and efficacy. Key words: Hepatitis B Serum Markers; Time-resolved Fluorescence Immunoassay; Fluorescence Quantitative PCR Technique; Clinical Application