Influence of Hypoxic Condition on Invasion of Cultured Trophoblast / 대한주산의학회잡지

OBJECTIVE: To investigated whether lowering oxygen tension affects invasion of cultured trophoblast. METHODS: Trophoblasts were isolated from the normal placenta in early pregnancy(6-10 weeks in gestation). Isolated trophoblasts were cultured under normoxic(5% CO2, 95% humid air in incubator) and hypoxic(MERCK, 1% O2, 99% CO2) conditions for 24, 48 and 72 hours, respectively. The proliferation ability was measured using [H3] thymidine assay. Total RNA was extracted from the cultured trophoblasts. The expressions of matrix metalloproteinase(MMP-2) and tissue inhibitor of metallo- proteinase(TIMP-2) were determined by reverse transcription- polymerase chain reaction(RT-PCR) and Northern blot analysis. The invasiveness of cultured trophoblast was observed using in vitro invasion assay. RESULTS: [H] thymidine assay indicated that cellular DNA synthesis was not affected by the culture condition. The expression of MMP-2 mRNA was decreased at 24 hours and then progressively increased in the time-dependent manner in each culture condition. The expression of TIMP-2 was decreased in the time-dependent manner under hypoxic condition. In vitro invasion assay revealed that the cultured trophoblasts under hypoxic condition has more invasive ability than them under normoxic condition. CONCLUSION: These data suggests that hypoxic condition may stimulates the invasion of trophoblast in the human placentation. And MMP-2 and TIMP-2 may be related to control their invasiveness under hypoxic condition.

Separation of invasive subpopulation from primary human renal cell carcinoma via in vitro invasion assay / 第二军医大学学报

Objective:To isolate the invasive and non-invasive cells from primary human renal cell carcinoma(RCC) in vitro.Methods: Fresh RCC surgical specimens from 32 primary RCC patients were primarily cultured following enzyme digestion or mechanical minimization in vitro.In vitro invasion assay using the Transwell cultures coating Matrigel was performed for separation and recovery of invasive and non-invasive cells from the primary culture of 3 RCC patients.The concentration of Matrigel,recovery time and trypsinization were subsequently optimized.Results: The successful rate of primary culture was(90.6%)(29/32).Recovery of invasive cells was performed ideally when matrigel(diluted into 1.0 mg/ml and 20 ?l) was coated onto the filter of the well;cell suspension was at a concentration of 5?10~(5)/ml and invasive cells were recovered on the 5th day of culture.The growth of non-invasive cells was scattered,while that of the invasive cells was focal.The doubling time of invasive cells was 36.1 h and that of non-invasive was 50.6 h.Conclusion: The in vitro invasion assay using the Transwell is able to separate and recover the highly invasive primary RCC cells.The primary cells represent intact subpopulation composition,but it can hardly get through the life span of human primary tumor cells.