Establishment And Characterization Of A New Primary Human Renal Carcinoma Cell Line

[Background]RCC is one of the most common malignancies in genitourinary system. Though the RCC morbidity is approximately 3% of all body malignancies morbidity , recently it is keep growing by 2% year after year, every year more than 100,000 people are died of RCC .In China, RCC morbidity is in the 2nd position in genitourinary system carcinomas, just behind of Bladder Cancer, and it is keep growing faster especially in recent years, which makes RCC becomes one of the most fatal carcinomas to people health. Most of RCC has Congenital Multidrug Resistance, and not sensitive to chemotherapy and radiotherapy,surgery is only effective to the patients who are in the early stage of RCC, for most patients with Intermediate stage RCC and terminal RCC , there is no special therapy which is effective. About 50% of patients are found in the Intermediate stage RCC and terminal RCC when going to hospital, around 40% patients'RCC metastasis or recurrence. The expected life is short for patient who has RCC metastasis, the average time is no more than one year, the chance of patients living more than 3 years is lower than 5%. That is why, researching on RCC pathogenesis and effective therapy has become the most important direction for Urology.In 1988, China mainland build up the 1st vitro Human RCC Cell Line[4], which contributed a very important cell model to RCC basic research and clinical study。However, by the increase of generations in vitro, special vitro biological characteristics may change gradually or disappear. That's why keep on establishing new biological system has already become the important piece of researching on RCC pathogenesis and effective therapy.Nude mice is treated as the most suitable carrier when establishing Human carcinoma animal model due to it's immunodeficiency characteristics. From being discovered in 1962 by Crist from UK, to 1969 Rygaard achieved the 1st success in repotting human Colonic Adenocarcinoma to the inside of nude mice, till now on there are more than 400 human carcinoma nude mice repotting model. In China, after the nude mice are being imported from 1979, there have already been more than 10 different kinds of human carcinoma model. The nude mice have been used in Oncology,Microbiology,Immunology,Pathology and the research of every other different preclinical medicine, which is the ideal instrument to researching malignancies pathogenesis and biological characteristics.Inoculating tissue mass in hypodermis of nude mice is a normal way to establish carcinoma cell system.. The results is better than culturing tissue mass directly and injecting a tumor cell suspension. BALB/cnu/nu nude mice can minimize Immunological rejection chances because of having normal B CELL, NK CELL but T CELL limitation inside the body. Based on the measures above, the research in this paper successfully established a stably passaged RCC Cell Line[Objective]To establish a nude mice transplant model by inoculating RCC patient surgery specimen into nude mice body, and establishing a new human RCC cell line by nude mice passages and vitro passages, then assaying the cell line, contributing a experiment model for RCC basic research for further research.[Materials and methods]1. Establishing Human RCC Primary Cell LineFrom 2008 to 2009,fresh surgical samples of RCC were obtained from 20 patients;each specimen was cut every four cases of 0.5 cm×0.5 cm×0.5 cm tissue and inoculated subcutaneously into the right posterior leg and the back of two nude mice within 1h after surgery. After three passages in the nude mice,the tumor tissue was cultured in vitro. Continuingly passage 110 times.2. Biological Characteristics Assay on RCC Primary Cell LineObserve the cell morphology and structure, draw the Cell growth curve, determination of cellular DNA content,analysis Karyotype,the experiment of the colony formation ability,the experiment of (Con A)concentration,determination of the biological activity of IL-6 and the histologic analysis.[Results]1. Establishing Human RCC Primary Cell LineIn which one of nude mice'subcutaneous tumor has grown.continued passage, the tumor'growth significantly faster. Tumor specimens were obtained in vitro and culture, continuous transmission of 110 generations, the human renal cancer cell line of XJG-9201 was got.2. Biological Characteristics Assay on RCC Primary Cell LineThe Cell morphology and structure, differentiate degree are consistent with primary carcinoma; chromosome mode is 65; the Population Doubling Time of cells is approximately 38.2h; According to the Cell Cycle Analysis, G1 period is 62.7%, G2 period is 11.2%, S1 period is 25.3%; the colony formation ability on soft agar is 70%; when the concanavalin A (Con A)concentration is 12.5ug/ml, the cell obviously agglutinate; The histologic analysis has the characteristics of Renal Clear Cell Carcinoma.[Conclusions]1. Successfully establishing RCC Primary Cell Line XJG-92012. The RCC Primary Cell Line XJG-9201 has the same Biological Characteristics as the primary carcinoma, continuous passages 115 times in vitro for more than 1 year. The Cell morphology didn't change, the Growth Cycle is steady, already become a stable cell line.