| Objective:This subject in the study of estrogen (ER) receptor, and progesterone receptor (PR),μopioid receptor (MOR) in the human trophoblast cells, based on , to interfere in vitro with morphine in cultured human trophoblast cells in early pregnancy were observed trophoblast ERmRNA, PRmRNA, MORmRNA expression changes, through the study of exogenous opioids on the trophoblast cells directly, in order to further seek to reduce or block the morphine dependence on the negative effects of pregnant women and fetus to provide a theoretical basis for further study opiate dependence and withdrawal symptoms provide the basis for the cause and treatment of and ideas.Materials and Methods:Specimens from December 2008 to March 2009 the First Clinical Hospital of Shanxi Medical University, Obstetrics and Gynecology flow chamber 6 to provide a 10-week pregnant abortion placental villi in 10 cases, and with the patient's informed consent. The health of pregnant women without complications. Age 20-30 years old, B super-confirmed intrauterine pregnancy. Villi obtained immersed in culture medium,4℃preservation, using modified method were isolated and cultured.Will train out of trophoblast cells by cytokeratin CK-7 (cytokeratin 7) antibody, vimentin (viment) antibody immunohistochemical staining, and the aggregation of the enzyme reverse transcriptase chain reaction (RT-PCR) method for identification; reference to the relevant literature to the cultured cells were divided into six sections, namely the control group (blank control); morphine low dose group (simply by adding morphine, the final concentration of 10-7 mol·L-1); morphine low dose group (simply by adding morphine, the final concentration of 10-6 mol·L-1); morphine dose group (simply by adding morphine, the final concentration of 10-5 mol·L-1); morphine high dose group (simply Join morphine, the final concentration of 10-4 mol·L-1); morphine+sodium casein ketone group (one in advance by adding sodium casein incubated 1h, then add morphine, both of the final concentration of 10-5 mol·L-1). Using RT-PCR method, using samples of gray andβ-actin ratio, calculated for each experimental group ERmRNA, PRmRNA, MORmRNA expression.All data are based on all the data to mean±standard deviation (±s) that the use of statistical software SPSS13.0 the results of RT-PCR single-factor analysis of variance and LSD test to compare dose-effect relationship between logarithmic linear correlation analysis; toα=0.05 level for the test. Result:(A) The first exchange of medium, the sides of the bottle attached to the organization of a large number of small blocks, evenly spread, there are more triangular or polygonal cells, spreading out along the edge of tissue surrounding the formation of multi-layered tissue outgrowth organization there are many large blocks of triangular or irregular cell adhesion, and everywhere there are 6-20 cells formed around the cell colony; cultured 4-6 days cell growth is very rapid, outgrowth increased gradually, the cell density increases, some growth of tissue cells between the large woven together to form a single layer of the cultured cells. CFU gradually increasing integration into the film, the cell gap to narrow, cobblestone was changed, in 6-8 days cells can be subcultured. Inverted microscope can be seen uniformly distributed human chorionic trophoblast cells were irregular polygon, oval nucleus is located near the center of the cytoplasm, abundant clear cytoplasm. Cells of epithelioid cell morphology, spreading growth was flaky. Differential adhesion method using purified trophoblast cells, immunohistochemical staining showed that cultured cells purified by CK7 antibody staining, about 90% of the cytoplasm was stained brown to dark brown, no nucleus staining, Vimentin stain-negative cells accounted for the vast majority, a handful of vimentin staining brown positive signal for fibroblasts. RT-PCR assay showed the cultured cells MORmRNA. Description with the modified method can be easily from human trophoblast tissue to obtain a large number of high-purity human chorionic trophoblast cells, and has maintained its function in the body can be used for further study.(B) RT-PCR results showed that:ERmRNA, PRmRNA, MORmRNA and restricted reference materialsβ-actin gene products were in the 249bp,196bp,342bp,307bp occurring at the specific amplified bands. Different concentrations of morphine group ERmRNA, PRmRNA, MORmRNA expression with control group and morphine+sodium casein-one comparison between groups were statistically significant (P<0.001) and the concentration of morphine along with the decrease of human trophoblast cells ERmRNA, PRmRNA, MORmRNA the increased expression of a negative correlation between the two (r=-0.996, P<0.001; r=-0.983, P<0.001; r=-0.999, P<0.001).Conclusion:Human trophoblast cells exist inμ-opioid receptors; At the molecular level, morphine can directly makes trophoblasts ERmRNA, PRmRNA, MORmRNA expression decreased in a dose-dependent reduction. |
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