Cultures Of Human Amniotic Cells Research Articles

İnsan amniotik hücre kültüründe nikotin ve resveratrolün Sox2 ve Sox4 genlerinin ekspresyon seviyeleri üzerindeki etkilerinin real time PCR ile araştırılması

Purpose: The aim of this study was to show the effects of nicotine on expression levels of SOX2 and SOX4 genes, which are known for their master gene features, in human amniotic cell cultures. The study also aimed to show the effects of resveratrol, an antioxidant, on expression levels of these genes in human amniotic cell cultures, which are treated by nicotine.Material and Methods: Twenty patients were included in this study and for each patient; control, nicotine treated and nicotine + resveratrol treated cell culture groups are formed. The expression levels of SOX2 and SOX4 genes are examined in each group by using real time RT-PCR.Results: Change in expression levels of SOX2 and SOX4 genes in nicotine treated group were found to be statistically significant. Also, when groups treated with nicotine and nicotine + resveratrol were compared the difference was found to be statistically significant.Conclusions: Nicotine increased the expression levels of SOX2 and SOX4 genes by 60% in human amniotic cell cultures and resveratrol was found to be an important antioxidant that reduces the increased expression levels of SOX2 and SOX4 genes caused by nicotine treatment.

Construction of tissue-engineered skin by culture of human amniotic epithelial cells on human de-epidermized dermis for the repair of skin defects in nude mice: an experimental study

Objective To evaluate the feasibility of repair of full-thickness skin defects in nude mice with tissue-engineered skin which was constructed by culture of human amniotic epithelial cells (hAECs) and fibroblasts on human de-epidermized dermis (DED) . Methods Healthy human amniotic tissues were treated with trypsin at a low concentration in multi-steps to prepare hAECs, and a two-step collagenase digestion was used to treat healthy children′s prepuce tissues to prepare fibroblast suspensions. When fibroblasts were cultured in vitro up to passage 3-5 and hAECs up to passage 2, they were seeded on the reticular dermal surface and basement membrane surface of the DED respectively to construct the tissue-engineered skin. A total of 20 healthy male nude mice aged 3-4 weeks were enrolled into this experiment, and full-thickness skin defects were made on the middle of the back of mice. Then, these mice were randomly divided into 2 groups by using a lottery method, and reconstructed full-thickness tissue-engineered skin grafts and vaseline oil gauze were used to cover the wounds in the tissue-engineered skin group and control group respectively. The whole body and transplantation sites of the nude mice were observed on day 7, 14, 21 and 28 after transplantation, the wound healing time and rate were compared between the above two groups, and skin tissues at the transplantation site were harvested at 4 weeks after transplantation and subjected to histological examination. Results HAECs had stem-cell characteristics and expressed octamer-binding protein-4 (OCT-4) and embryonic marker stage-specific embryonic antigen-4 (SSEA-4) . After 2-week organ culture, the in vitro reconstructed tissue-engineered skin showed 4-9 continuous layers of stratified epithelium, and the histological structure of the epidermis was similar to that of the normal human skin. Compared with the control group, the tissue-engineered skin group showed significantly higher wound healing rates on day 7, 14 and 21 after transplantation (57.49% ± 6.11% vs. 22.93% ± 4.26%, 92.80% ± 3.10% vs. 54.57% ± 7.94%, 98.83% ± 0.25% vs. 91.16% ± 4.79%, respectively; n= 10, t= 27.36, 32.23, 11.80, respectively, all P < 0.001) , shorter wound healing time[ (21.51 ± 1.51) d vs. (28.80 ± 1.14) d, n= 10, t= 42.23, P < 0.001], with the color of skin grafts closer to that of autologous skin on day 28 after transplantation. Histological examination revealed distinct stratification of the epithelium, obvious keratinization and favorable growth of cells in the dermis in the tissue-engineered skin group, but thin epithelium with some defects, indistinct stratification of the dermis, and inflammatory cell infiltration in the control group. Conclusion Tissue-engineered skin constructed by the culture of hAECs and fibroblasts on human DED can survive in nude mice after transplantation, resulting in a more favorable healing of wounds, and is expected to serve as a kind of ideal tissue-engineered skin. Key words: Tissue engineering; Epithelial cells; Dermis; Skin transplantation; Wound healing; De-epidermized dermis; Human amniotic epithelial cells

Culture of human amniotic cells: A system to study interferon production

This study investigated whether primary culture of human amniotic membrane cells (PCHAM) could be used as an in vitro model system for the study of interferon (IFN) production. PCHAM cells infected with Newcastle disease virus (NDV) produced the two antigenic types of IFN, previously shown in a amniotic membrane cells (HAM) system. PCHAM IFN was detected as early as 2 h after NDV infection and was composed by two antigenically distinct fractions, one neutralized with anti-HuIFNβ antibody and another that is not related to IFNβ, -α and -γ. These fractions correspond respectively to 80 and 20 per cent of the IFN produced 4 h after virus induction, 55 and 45 per cent of the IFN produced from 4 to 12 h and 67 and 33 per cent of the IFN produced 12 h after virus induction. A cDNA library, established from PCHAM with or without NDV infection, was screened for IFNα and −β using specific primers. The PCR product, amplified by IFNβ primers, was cloned, sequenced and expressed in Escherichia coli M15. The sequences of several cloned cDNAs were identical to HuIFNβ gene and the antiviral activity of the expressed protein was neutralized only by antlHuIFN-β antibody. The other IFN fraction not neutralized by polyclonal antibodies anti-IFNβ, -α and -γ is now being studied.

Effects of ascorbic acid on Chlamydia trachomatis infection and on erythromycin treatment in primary cultures of human amniotic cells.

Ascorbic acid (vitamin C) is an essential nutrient for humans. It may also be needed by Chlamydia trachomatis, an intracellular bacterium. We investigated the effects of vitamin C on the growth of C. trachomatis E/UW-5/Cx in a primary culture of human amniotic epithelial cells. The results showed that vitamin C enhances C. trachomatis infection at concentrations of 0.2, 0.6, and 1.2 mg/dl (P less than 0.001). These three concentrations represent the in vivo concentrations of deficiency, normal, and overload levels in serum, respectively. The enhancement was dose dependent. However, the growth of C. trachomatis was inhibited at vitamin C concentrations of 120 and 1,200 mg/dl. The inhibitory effect of erythromycin against C. trachomatis was shown to be reduced in the presence of vitamin C at the three concentrations tested (P less than 0.025-0.001), and MICs were four times greater (1.6 versus 0.4 micrograms/ml). Human amniotic cells were tolerant to vitamin C concentrations of up to 1,200 mg/dl. The results show that vitamin C may be an important nutrient for C. trachomatis and that incorporation of vitamin C in the culture medium may enhance the isolation and propagation of C. trachomatis in cell cultures.

Cinematographic observations of growth cycles of Chlamydia trachomatis in primary cultures of human amniotic cells

Time-lapse cinematography was used to study the growth cycle of Chlamydia trachomatis in primary cell cultures of human amnion. Twelve preterm and twelve term placentas were obtained within 8 h of delivery, and epithelial cells were dissociated from the amniotic membranes by trypsinization and grown in Rose chambers. The epithelial nature of the cultured cells was documented by morphology and by immunofluorescence staining for cytoskeletal proteins, which matched the staining of intact amnion. With regular feedings, uninfected cultures remained healthy for up to 30 days. Confluent cultures (7 to 10 days) were infected with a genital strain (E/UW-5/CX) of C. trachomatis at 10(5) infectious units per chamber. Infections were done in culture medium without cycloheximide, which is often used to induce susceptibility of the cells. Between 66 and 90% of the cells were infected. Intracytoplasmic inclusions were visible by 18 h post infection (p.i.) and grew larger as the organisms inside multiplied. By 72 h p.i., the inclusions occupied the entire cytoplasm of the host cells. Further growth of the inclusions overdistended and ruptured the host cells on days 3 to 7. Cells not infected by the original inoculum became infected on day 5 or 6 p.i. by the chlamydial particles released from the ruptured cells. No amniotic cell was ever observed to survive the infection. The data presented support the hypothesis that amniotic epithelium is susceptible to infection and damage by C. trachomatis. This culture system provided detailed and dynamic observations of chlamydial infection under conditions more nearly physiologic than previously reported.

Enzyme activation of human prolactin: a potential basis for a bioassay

Small cultures of human amniotic cells were preincubated for 24 h. Human prolactin was then added to the medium. After a further short period of incubation the tubes were chilled, the medium removed and the cells rinsed with saline. The tubes then received cold Tris-sucrose and were frozen, to disrupt the cells. After thawing, adenosine triphosphatase (ATPase) and p-nitrophenyl phosphatase (PNPase) were measured. Buffer was added containing either ATP or PNP and the tubes were incubated for 30 min. Inorganic phosphate released from ATP and p-nitrophenol was measured spectrophotometrically. Prolactin stimulated both enzyme activities. The ATPase log dose-response curve was linear between approximately 12.5 and 200 mIU/l. It was inhibited by ouabain. Isobutyl-1-methylxanthine inhibited the ATPase but not the alkaline phosphatase activity. One of these human amniotic cell enzymes may provide the basis for a sensitive bioassay for human prolactin.

In Vitro Angiogenic Activity of RNA From Leukemic Lymphocytes

RNA was extracted from leukemic lymphocytes by a fluorocarbon method, and primary cultures of human amniotic cells were exposed to the lymphocyte RNA. Angioid tubular and cordon-like structures in branching out and criss-cross patterns with budding ends developed in these cultures within 7 to 12 days of the first exposure. Although there was no opportunity to study preparations from nonleukemic lymphocytes, extracts of normal peripheral blood cells and blood rich in granulocytes had no angiogenic effects. In view of the recent renewal of interest in tumor and tissue angiogenic factor as well as in angiogenic activity by transformed lymphocytes, in addition to the intriguing angioblastic component of Lukes-Rappaport immunoproliferative syndromes, this work may point to the ribonucleic acid nature of the vessel-forming principle. Various cell-differentiating properties of RNA preparations have been repeatedly described, and thus it is not altogether surprising that lymphocyte angiogenic factor may originate in RNA.

Prolonged Growth of Leishmania Species in Cell Culture

Five strains of Leishmania, two of L. donovani, and three strains from human cutaneous lesions (L. mexicana, L. braziliensis, and L. braziliensis pifanoi), have been maintained for at least a year with apparent multiplication in the intracytoplasmic leishmanial stage in a dog sarcoma cell line. When temperature of incubation was 36 C, L. donovani were leishmaniform and flagellates were absent. At 33 C, extraand intracellular parasites were elongate and flagellates were numerous. With the "cutaneous" strains leishmaniforms were present at 33 C and flagellates appeared at 28 C. At 36 C, parasites of the two strains of L. braziliensis disappeared but those of L. mexicana survived. Leishmaniform infections of cultured cells with "cutaneous" strains were further characterized by large numbers of parasites within cytoplasmic vacuoles, while infections with L. donovani were characterized by relatively few parasites apparently embedded in the cytoplasm. Many types of cell culture have been infected with Leishmania spp., although continuous cultivation of intracellular leishmanial forms in tissue cultures has been an elusive goal. In this laboratory, for example, human amniotic cells were readily infected, but the intracellular leishmaniforms eventually disappeared from the cultures (Frothingham and Lehtimaki, 1967). However, continuous intracellular growth in vitro in the leishmanial form was described by Lamy and co-workers (1964), who studied L. donovani in a cell line derived from a dog's sarcoma. We report here on the use of this cell line for prolonged culture of intracellular leishmaniforms of two strains of L. donovani and of three strains of Leishmania derived from patients with cutaneous disease. MATERIALS AND METHODS Details of most of the methods and of the origins of some of the parasites have been described (Frothingham and Lehtimaki, 1967). L. donovani: Strain P, isolated by Dr. S. C. Pan from a Pakistani with visceral infection, was maintained in NNN cultures for more than a decade; cell culture passages were initiated with leptomonads. Strain S, from a Sudanese with visceral infection, was isolated by Dr. Harry Browne and maintained in hamsters until infection of cell cultures was initiated with infected ground splenic tissue. Strain G, from a dog with visceral disease, probably acquired in Greece (Theran and Ling, 1967), was isolated by us and maintained in hamReceived for publication 7 June 1968. * Supported in part by Research Grant AI-01023 and Training Grant AI-177 from the NIAID, U. S. Public Health Service. sters until infection of cell cultures was initiated with ground spleen. Other Leishmania spp.: Three strains originated from patients with cutaneous disease. L. mexicana, a strain described by Garnham and Lewis (1959), was furnished by Dr. Marshall Hertig who had maintained it for several years in hamsters; infection of cell cultures was started with flagellated forms from our 26th NNN passage. L. braziliensis pifanoi, from a patient with difusa ype of cutaneous leishmaniasis, was furnished by Dr. J. Convit. Inoculum for the present experiments with this strain was derived from nutrient fluid and cells scraped from infected cultures of human amniotic cells (Frothingham and Lehtimaki, 1967). L. braziliensis (sensu lato) was isolated by Dr. Bryce Walton in 1965 from a cutaneous lesion on a North American infected in Panama. Infection of cell cultures with this strain was initiated with flagellated forms from the 19th passage in NNN. The dog sarcoma cell line, kindly supplied by Dr. Lamy, was maintained at 36 C by conventional methods in stationary bottles. Cultures were nourished with Parker's medium 199 containing 10% fetal bovine serum (FBS) previously heated at 56 C for 30 min. For experiments, cells were grown on coverslips in Leighton tubes in nutrient medium containing 5 to 20% fetal bovine serum. Nutrient medium was changed twice a week. Incubation was at 28, 33, or 36 C ? 1 C. Cultures were examined microscopically, and cover slips were studied after fixation with Zenker's acetic acid and staining in Giemsa's. The percentage of cells infected was determined as already described (Frothingham and Lehtimaki, 1967). The average number of organisms per infected cell was determined by counting the number of parasites in 100 consecutively encountered infected cells. The number of cells per culture was calculated from the number of cells in an apparently representative area of the cell sheet approximating 1/450th of the total area. The number of intracellular organisms per culture was calculated from all three foregoing values.

Leishmania in primary cultures of human amniotic cells.

Leishmania in primary cultures of human amniotic cells.

Shortening by Actinomycin D of Latent Period of Multiplication of Sendai Virus

IT has been shown earlier that actinomycin D inhibits the multiplication of influenza viruses without any influence on Newcastle disease virus and Sendai virus multiplication1,2. It was found that the dynamics of Sendai virus reproduction in actinomycin-treated cultures of chicken fibroblasts and human amniotic cells were changed after the latent period of multiplication was shortened by 2 h (Fig. 1). To clarify this problem an autoradiographic investigation was carried out. Cultures of human amniotic cells infected with Sendai virus (1,000 ID50 per cell) were held at 35° C in Medium 199 or in this medium with 0.5 γ/ml. of actinomycin D. 30 min, 1, 2, 3 and 5 h after onset of incubation samples of cultures were exposed to 1 µc/ml. of tritiated uridine for 30 min, washed in BBS containing excess of unlabelled uridine, fixed in Carnoy solution, covered with emulsion type M and exposed for 1 month. Developed autographs were stained by the Unna method.