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In Vitro Separation of High Invasive and Low Invasive Subpopulation Cells from Human Renal Cell Carcinoma

Author: ChenLei
Tutor: YuDeXin
School: Anhui Medical University,
Course: Surgery
Keywords: Renal cell carcinoma Cell separation Invasiveness Cell culture
CLC: R737.11
Type: Master's thesis
Year: 2008
Downloads: 48
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Abstract

Renal Cell Carcinoma occurs more commonly in urinary system, which is associated with more aggressive and extensive metastatic characteristics. One third patients have been in advanced stages when being detected for the first time,and forty percent patients reoccur or metastasize after operatiaons with worse prognosis. Renal Clear Cell Carcinoma is first most common subtype of Renal Cell Carcinoma. Therefore,developing a series of metastatic associated biological marks will be of great significance in detecting RCC effectively with the potential to reduce the death rate. It is critical step for screening and identifying metastatic associated biological mark that Renal Cell Carcinoma cell lines with different metastatic potential is established in the same genetic background.According to tumor heterogeneity theory:neoplasms contain subpoppulatiopn of cells having different metastatic potential,only those tumor cells can establish a metastasis which are capable of completing the entire process:dissociating from the primary mass,migrating through the interstitial extracellular matrix,invading from the basement membrance underlying the vascular endothelial cell layer and basement membrance to enter the interstitial matrix and establish a metastasis.Therefor,invasive and metastatic associated factors can be used as basis for isolating tumor cells with different invasive, metastatic potentials.Malignant cells must traverse basement membrances during their migration to sites distant from the primary tumor.Matrigel is a solubulized basement membrance preparation extracted from the Engelbreth-Holm-Swarm mouse sarcoma,a tumor rish in ECM proteins.Its major component is laminin,followed by IV collagen,heparin sulfate proteoglycans,and entactin.At room temperature, Matrigel polymerizes to produce biologically active matrix material resembling the mammalian cellular basement membrane ,which are thin continuous sheets applied over filters in transwell insert,blocking non-invasive cells from migrating through the membrane.In contrast, invasive tumor cells are able to detach themselves from and invade through the Matrigel and the 8 micron membrane pores.Therefor,it is well suited for selecting differently invasive cell phenotypes in response to a chemoattractant. Then we extract total RNA from high invasive and low invasive renal cell carcinoma cells respectively and analyze them.At last we use SMART to synthesis cDNA.Objective : To isolate the high invasive and low invasive renal cell carcinoma cells from human renal cell carcinoma (RCC) cell line ACHN in vitro, extract total RNA from high invasive and low invasive renal cell carcinoma cells respectively and synthesis cDNA.Methods: The renal cell carcinoma cell line ACHN was serial subcultivation in vitro. Then in vitro invasion assay using the Transwells coating Matrigel were performed for separation and recovery of high invasive and low invasive cells from the serial subculture of cells. The concentration of Matrigel, trypsinization and recovery time were subsequently optimized.Then we extract total RNA from high invasive and low invasive renal cell carcinoma cells respectively and analyze them, then we use SMART to synthesis cDNA.Results: Matrigel (diluted into 1.17mg/ml) was coated onto the filter of the Transwell; cell suspension was at a concentration of 5×10~5/ml and invasive cells were recovered on the 48h of culture. When all above were prepared well, the recovery of invasive cells was performed ideally. The growth of non-invasive cells was sporadic, while that of the invasive cells was accumulative. Total RNA extracted from high invasive and low invasive renal cell carcinoma cells are good. SMART can be used to synthesis cDNA with a small quantity of RNA.Conclusions: In vitro the Transwell is able to quickly separate, recover and culture the highly invasive RCC cells. At the same time, the cell line ACHN had a nice representation,and it can easily go down to the future generation. we successfully extract total RNA from high invasive and low invasive renal cell carcinoma cells respectively and synthesis cDNA, which lay a substantial fundament for researching metastatic associated genes.

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