Time Fluorescent Polymerase Chain Reaction Research Articles

Effects of human umbilical cord mesenchymal stem cells-derived exosomes on fracture healing in rats through the Wnt signaling pathway.

To investigate the role of human umbilical cord mesenchymal stem cell (hucMSC)-derived exosomes in the Wnt signaling pathway and their effects on fracture healing in rats. A total of 24 healthy male Sprague-Dawley (SD) rats were randomly divided into 3 groups, of which the experimental groups were injected with Phosphate-Buffered Saline (PBS) and hUCMSC-derived exosomes, respectively, at the fracture site, and a blank control group was set. At 2 and 3 w after treatment, respectively, the healing condition at the fracture site in the rats was detected by micro-computed tomography (CT). The protein expressions of β-catenin and Wnt3a of the Wnt signaling pathway in the bone tissue were measured via Western blotting (WB) assay. Quantitative Real Time-fluorescence Polymerase Chain Reaction (qRT-PCR) was performed to determine the expressions of osteogenic marker genes [collagen type I (COL-1), osteopontin (OPN) and runt-related transcription factor 2 (RUNX2)]. The results of the micro-CT scan showed that the rats treated with exosomes had better apposition of the fracture site, and the appearance of cortical bone was continuous. The fracture sites in the blank control group and PBS injection group were not healed, and the appearance of cortical bone was discontinuous, with significant fracture lines. According to the WB results, the protein expression levels of β-catenin and Wnt3a in exosome treatment group were significantly higher than those in the blank control group and PBS injection group (p<0.01). The qRT-PCR results indicated that the expression levels of COL-1, OPN and RUNX2 in exosome treatment group were increased evidently compared with those in the other two groups (p<0.01). HucMSC-derived exosomes are probably involved in the repair of fracture in rats through the Wnt signaling pathway.

DSCAM-AS1 promotes tumor growth of breast cancer by reducing miR-204-5p and up-regulating RRM2.

Breast cancer (BC) is a common malignancy worldwide. More than 3 700 000 women die of BC every year. DSCAM-AS1 was overexpressed several kinds of cancer and miR-204-5p was lowly expressed, which indicated that miR-204-5p had anti-tumor activity and DSCAM-AS1 had pro-tumor activity. We intended to analyze DSCAM-AS1, miR-204-5p, and ribonucleotide reductase M2 (RRM2). Microarray analysis and quantitative Real Time fluorescence Polymerase Chain Reaction (qRT-PCR) were employed to determine DSCAM-AS1 and miR-204-5p expression. Luciferase reporter assay was applied to examine the target relationship between DSCAM-AS1, miR-204-5p, and RRM2. Cell Counting Kit-8 (CCK-8 assay), transwell assay, and flow cytometry were used to detect cell proliferation, invasion, and apoptosis. The expression of DSCAM-AS1, miR-204-5p, and RRM2 were confirmed by Western blot. We also conducted in vivo assay to verify the effect of DSCAM-AS1. DSCAM-AS1 was up-regulated, while miR-204-5p was down-regulated in BC tissues and cells. DSCAM-AS1 directly targeted miR-204-5p. DSCAM-AS1 promoted the proliferation and invasion of BC cells by reducing miR-204-5p and inhibiting miR-204-5p expression. DSCAM-AS1 expression was related to the expression of RRM2, and miR-204-5p could reverse the function of DSCAM-AS1. RRM2 was up-regulated in BC cells, and miR-204-5p inhibited RRM2 expression by targeting RRM2. Overexpression of RRM2 stimulated proliferation and cell invasion and impeded apoptosis. In vivo experiments showed that knockdown of DSCAM-AS1 decreased the tumorigenesis of BC cells, increased the expression of miR-204-5p. DSCAM-AS1 promoted proliferation and impaired apoptosis of BC cells by reducing miR-204-5p and enhancing RRM2 expression. DSCAM-AS1/miR-204-5p/RRM2 may serve as novel therapeutic targets for BC.

English

&nbsp; To investigate the&nbsp;intercellular adhesion molecule-1&nbsp;(ICAM-1) expression in the liver and serum hepatitis B virus (HBV) DNA level in chronic hepatitis B (CHB) patients before and after interferon (IFN) treatment, a total of 51 CHB patients were recruited and treated with IFN-&alpha; 1b for 6 months. The expressions of ICAM-1 and serum HBV DNA were determined at baseline and after 6-month treatment by using immunohistochemistry and real time fluorescence polymerase chain reaction (PCR). The expression of ICAM-1 in the patients with moderate to severe CHB was markedly higher that that in mild CHB patients (P&lt;0.05) at baseline and after 6-month treatment. Furthermore, IFN treatment could dramatically decrease the ICAM-1 expression among patients with moderate to severe chronic hepatitis B (CHB) (P&lt;0.05). However, the expression of ICAM-1 was relatively stable in patients with mild CHB even after 6-month treatment (P&gt;0.05). In addition, there was a positive correlation between serum level of HBV DNA loads and ICAM-1 expression on hepatocytes in CHB patients, among patients with higher ICAM-1 expression (++, +++, ++++), the serum level of HBV DNA was markedly lowered by IFN-&alpha;1b treatment (P&lt;0.05), which was not observed in patients only mildly positive for ICAM-1 (+). The results indicate that IFN treatment can down-regulate the expression of ICAM-1 in the liver of CHB patients and promote the clearance of HBV, which was more obvious in patients with higher ICAM-1 expression. &nbsp; Key words:&nbsp;Interferon, hepatitis B, intercellular adhesion molecule-1.

Most commonSLC25A13mutation in 400 Chinese infants with intrahepatic cholestasis

To establish the real time fluorescence polymerase chain reaction (RT-PCR) with dual labeled probes for fast detection of SLC25A13 gene mutation 851del4. Four hundred infants (< 1 year of age) with unexplained intrahepatic cholestasis from 18 provinces or municipalities in China were enrolled in this study for detecting their SLC25A13 gene mutation 851del4. Suitable primers and fluorescence-labeled probes for detecting SLC25A13 gene mutation 841del4 were designed. Normal and mutant sequences were detected by PCR with two fluorescence-labeled probes. After a single RT-PCR, results were obtained by analyzing the take-off curves. Twenty-four positive and 14 negative samples were retested by direct sequencing. Eight homozygous and 30 heterozygous mutations were detected in 46 mutant alleles with a 851del4 mutation rate of 5.8% (46/800). Twenty-six and 20 mutant alleles were observed respectively, in 474 and 242 alleles from the intermediate and southern areas of China. No mutant allele was detected in 84 alleles from northern China. Twenty-four positive samples including 4 homozygous and 20 heterozygous mutations, and 14 negative samples were retested by direct sequencing, which confirmed that the accuracy of RT-PCR was 100%. RT-PCR can detect the mutation 851del4 in infants with intrahepatic cholestasis with an accuracy of 100%.

Possible mechanisms of Cyclosporin A ameliorated the ischemic microenvironment and inhibited mitochondria stress in tree shrews’ hippocampus

Objective: The ischemic brain damage is always accompanied by the significant accumulation of glutamate and calcium ions (Ca 2+). Our objectives were to observe the effects of glutamate and Ca 2+ overloading in tree shrew's hippocampal microenvironment on mitochondrial stress resulting in cytochrome C release and caspase apoptotic gene activation, and to explore the possible mechanism of Cyclosporin A (CsA) inhibiting mitochondrial stress. Methods: The thrombotic focal cerebral ischemia was induced by photochemical reaction in tree shrews. The extracellular contents of amino acidic neurotransmitters and Ca 2+ were determined, respectively, with high performance liquid chromatography (HPLC) and atomic absorption spectrophotometry at 4, 24 and 72 h after cerebral ischemia. The glutamate–calcium chloride solutions were microperfused into hippocampus by a kind of single-pumped push–pull perfusion (SPPP) system under three-dimensional orientation instrument in tree shrews. At 24 h, the expression of cytochrome C was observed in perfused lateral hippocampus by immunochemistry. Also, the hippocampus was removed, then mitochondria and cytoplasmic fragment were divided by low temperature centrifugation and the distribution of cytochrome C was assessed through Western blot. Real time fluorescence polymerase chain reaction was used to evaluate the relative amounts of caspase-3 and caspase-9 mRNA. In the treated group, CsA (40 mg/kg) was intravenously injected at 6 h after the microperfuse or cerebral ischemia. The glutamate–calcium solutions were perfused into the hippocampus and inspected the above-mentioned items at 24 h. Data were compared between the two groups (ischemia group vs. sham group, or ischemia group vs. CsA group). Results: Thrombotic cerebral ischemia led to significant increase in extracellular glutamate and Ca 2+ level of hippocampus ( P < 0.01). The cerebral ischemia group and the microperfusion group, which cytochrome C immunoreactivity increased and Western blot analysis demonstrated that the cytochrome C content in the mitochondria of hippocampal cells decreased ( P < 0.01), but the cytochrome C in the cytosol increased ( P < 0.01). When CsA was intravenously injected at 6 h after the microperfusion or cerebral ischemia, the cytochrome C expression weakened and its release was diminished to a lesser extent. By real time PCR, in relation to the control group, the caspase-3 and caspase-9 mRNA was higher in the glutamate–calcium chloride solution perfused group. CsA treatment cut down the contents of caspase-3 mRNA and caspase-9 mRNA ( P < 0.01). Conclusions: It is a primary factor that glutamate and Ca 2+ accumulate in hippocampal microenvironment, which results in proapoptotic protein cytochrome C release from mitochondria into cytoplasm and caspase cascade activation, and finally mitochondria stress and neuronal secondary injury appear. The neuroprotection of CsA is in relation to inhibiting glutamate receptor overactivation and reducing the Ca 2+ influx, which can decrease cytochrome C release and caspase mRNA transition.

Detection ofMycobacterium aviumsubsp.paratuberculosisin Bovine Manure Using Whatman FTA Card Technology and Lightcycler Real-Time PCR

A modified forensic DNA extraction and real-time fluorescent polymerase chain reaction assay has been evaluated for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) in bovine fecal samples using primers and fluorescent resonance energy transfer (FRET) probes targeting the IS900 gene sequence of MAP. DNA was successfully extracted from manure samples by utilizing the Whatman FTA card technology, which allows for simple processing and storage of samples at room temperature. The FTA cards were washed and subjected to a Chelex-100 incubation to remove any remaining polymerase chain reaction (PCR) inhibitors and to elute the DNA from the FTA card. This isolated DNA was then subjected to direct real time fluorescent PCR analysis. Detection of MAP DNA from bovine fecal samples spiked with known concentrations of viable MAP cells was obtained. The detection limits of the assay was consistently found to be between 10(2) and 10(4) colony forming units [CFU]/g, with some samples containing as low as 10 CFU/g, yielding positive assay results. This cost-efficient assay allows reporting of results as early as 4 h after fecal collection, which can be particularly useful in highthroughput herd screening.

Cytotoxicity and altered c‐myc gene expression by medical polyacrylamide hydrogel

Medical Polyacrylamide Hydrogel (PAMG)has been used in plastic and aesthetic surgery for years. However, its safety is still in doubt in many countries. In the current research, first an approach, using high performance liquid chromatography (HPLC), to determine the amount of residual acrylamide monomer (AM) in the PAMG was presented. Then the cytotoxicity of PAMG was investigated using cell counting and methyl thiazolyl tetrazolium (MTT) assay. To explore the mechanism of this toxicity, normal human fibroblasts cultured in medium extracts were analyzed. Membrane changes and other related parameters were investigated using flow cytometry (FCM). Real time fluorescent polymerase chain reaction (real time PCR) was also introduced to determine the biological response of the fibroblasts. During this process, three representative genes (p53, beta-actin, and c-myc, which are tumor suppressor genes, housekeeping genes, and proto-oncogenes respectively) were selected for examination. Results indicated that a method based on HPLC is practical and simple for determining AM in PAMG. The detection limits can reach the desired ppb level, and so it can fully meet the requirements of the studies of PAMG. Polyacylamide Hydrogel inhibits the growth of human fibroblasts and may cause the apoptosis of human fibroblasts. Moreover, it can alter physical parameters such as the size and the granularity of these cells. Furthermore, these three genes have a relatively typical amplification plot and highly related, wide-range standard curves, and so this reaction system is definitely suitable for the semiquantification of these genes. PAMG induces the increase of the message ribonucleic acid (mRNA) expression of c-myc, while the p53 and beta-actin remain even. This change is not related to the concentration of AM in the gel and may be incited by other components in the extract of PMAG.

Human papillomavirus-positive tonsillar carcinomas: a different tumor entity?

Human papillomavirus (HPV) infections are thought to be one of the causal factors in the development of head and neck squamous cell carcinomas (HNSCC), particularly in tumors arising from the Waldeyer's tonsillar ring. We screened 98 carefully stratified HNSCC and different control tissues for the presence of HPV DNA by nested polymerase chain reaction (PCR) specific for genital- and Epidermodysplasia verruciformis (EV)-associated HPVs and by HPV16-specific single step PCR. Typing was performed by direct sequencing and/or sequencing of cloned amplimers. On average HNSCC showed rather low HPV DNA prevalences; 18% of the oral cavity cancers, 8% of nasopharyngeal cancers, 25% of hypopharyngeal cancers and 7% of laryngeal cancers were HPV DNA positive. In contrast, HPV sequences could be detected in 45% of the oropharyngeal cancers, particularly tonsillar carcinomas (58%). Tonsillar carcinomas were significantly more likely to be HPV positive than tumors from any other site ( P<0.001). All tonsillar cancers contained oncogenic HPV types, predominantly HPV16 (13 of 14; 93%). Unaffected tonsils were available from two of these patients, but both tested negative for HPV DNA. Furthermore, no HPV DNA could be found in tonsillar biopsy specimens from control groups. Localization and load of HPV DNA was determined in HPV16-positive tonsillar carcinomas, their metastases and in unaffected mucosa using laser-assisted microdissection and subsequent real time fluorescence PCR. We demonstrated that the HPV genome is located in the cancer cells, whereas the infection of normal mucosa is a rare event. Quantification of HPV16 DNA in samples of seven patients yielded viral loads from 6 to 153 HPV DNA copies per beta-globin gene copy and the load values in both locations were roughly comparable. These loads are comparable with data shown for other HPV-associated lesions. Statistical evaluation of data related to clinicopathological parameters showed a significant correlation of the HPV positivity of tonsillar carcinomas with tumor grading ( P=0.008) and alcohol consumption ( P=0.029). Taken together our findings show a preferential association of HPV DNA with tonsillar carcinomas. Furthermore our results argue for HPV-positive tonsillar carcinomas representing a separate tumor entity, which is less dependent on conventional HNSCC risk factors.