| Objective: To detect the expression of Tspan-5 in maternal-fetal interface and study the effect of Tspan-5 on the proliferation and migration of trophoblast cell, detecting the trophoblast cells secrete h CG concentration after target Tspan-5 gene silencing and changes of treatment effect to chemotherapeutic drugs methotrexate(MTX), and to explore the effect of Tspan-5 in the maintenance of normal pregnancy, and possibility of Tspan-5 as a effectively diagnostic marker of gestational trophoblastic disease, seeking a new treatment target for gestational trophoblastic disease.Methods: Chapter One:(1)We used RNAi silencing the Tspan-5 expression in human choriocarcinoma cell line JEG-3 first, and then used Western Blotting to detect the expression of Tspan-5, calculated the efficiency of interference.(2)Following, we detected the proliferation and migration of the JEG-3 cells before and after interference by CCK-8 test, colony formation and scratch assay.Chapter Two: By collecting the villus and decidua of normal early pregnancy, spontaneous abortion and tubal pregnancy(included Fallopian tube mucosa), to detect the expression levels of Tspan-5 through Western Blotting.Chapter Three:(1)Roche ECL automated immunoassay system was used to detect the medium supernatant h CG levels in each group.(2)And then we detected the proliferation of different groups after adding methotrexate.Results: Chapter One:(1)After silencing Tspan-5 gene, western Blotting results showed that: Tspan5/NC band gray value was highest, Tspan5/1005 was lowest, the relative expression level of Tspan-5 after interfering from low to high was Tspan5/1005 <Tspan5/796 <Tspan5/464 <Tspan5/NC, so we chose Tspan5/1005 as an interference group to finish follow-up experiment.(2) Outcomes of colony formation assay: Interference group(Tspan5/1005) clone number of JEG-3 cell was significantly reduced compared with the control group(Tspan5/NC)(148±23 vs 276±31, P<0.01). There was no significant difference between non-interference group and control group(328±39 vs 276±31, P>0.1).(3)Outcomes of CCK-8 test: The JEG-3 cell proliferation activity of non-interference group was the highest, the control group(Tspan5/NC) group's proliferation activity was slightly lower, however there was no significant difference(1.200±0.103 vs 1.185±0.022, P>0.05) between the two. Compared to the control group(Tspan5/NC), the JEG-3 cell proliferation activity of interference group(Tspan5/1005) was decreased significantly(1.185±0.022 vs 1.076±0.028, P<0.01).(4)Outcomes of scratch assay: In the absolute value of the scratch width 24 hours after scratching minus the scratche width 0 hour after scratching, the interference group(Tspan5/1005) was significantly less than the control group(Tspan5/NC)(1.27±0.03 cm vs 1.97±0.38 cm, P<0.05).Chapter Two:(1) Tspan-5 expression in villus of spontaneous abortion group was less than in the normal pregnancy group, but Tspan-5 expression in the decidua of both groups was no significant difference.(2)There were no significant difference of Tspan-5 expression in the villus and decidua between normal pregnancy group and tubal pregnancy group.(3) Tspan-5 expression in the Fallopian tube mucosa was no significant difference compared to that in the decidua of tubal pregnancy group.(4) Tspan-5 expression in the Fallopian tube mucosa of tubal pregnancy group was significantly higher than that in the decidua of normal pregnancy group(1.62 ± 0.47 vs 0.82 ± 0.15, P <0.01).Chapter Three:(1)Overall, the h CG concentration in the medium supernatant of each group was gradually increased with time. After culturing 72 h the h CG concentration of interference group(Tspan5/1005) was significantly higher than the control group(Tspan5/NC)(699.3±85.2 IU/L vs 507.0±130.2 IU/L, P<0.05), but no significant difference between non-interference group and control group(Tspan5/NC). There were no significant difference among the three groups in culture 0h, 24 h and 48 h.(2)After adding MTX, JEG-3 cells in each group were gradually decreased along with MTX action time, the active cells of interference group(Tspan5/1005) were significantly less than the control group(Tspan5/NC) at 24 h after adding MTX(0.817±0.039 vs 1.021±0.042, P<0.01), and were significantly reduced compared to interference group(Tspan5 / 1005) without adding MTX as well(0.817 ± 0.039 vs 1.066 ± 0.023, P <0.01).Conclusion:(1)In this study, we found that the proliferation and migration of choriocarcinoma cells were reduced after using RNAi technology to knockdown Tspan-5 expression.(2)Composite the results of this and our preliminary experiment, Tspan-5 mainly express in extravillous cytotrophoblasts and cytotrophoblast, and the quantity of Tspan-5 expression was positively related with proliferation, migration and invasion of choriocarcinoma cells, so there is hope for Tspan-5 as a marker to diagnose and monitor gestational trophoblastic disease.(3)Tspan-5 expresses in the maternal-fetal interface, which may relate to the occurrence of abortion.(4)Tspan-5 also expressed in the Fallopian tube mucosa, and Tspan-5 expression of mucosal when tubal pregnancy was higher than that in normal early pregnancy decidua, whether to prompt the tube mucosaare more proliferative / adhesive than normal decidual cells when tubal pregnancy? That still need further study.(5)After silencing Tspan-5 gene, treated choriocarcinoma cells with chemotherapy drugs MTX can quickly achieve a therapeutic effect,, and the JEG-3 cell proliferation activity was rapidly inhibited. So Tspan-5 is expected to become the new target in clinical treatment of gestational trophoblastic disease. |
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