Establishment And Identification Of Malignant Pleural Effusion-derived Lung Cancer Primary Cell Culture And Its Chemosensitivity

BackgroundLung cancer is a major factor that directly impact the health of human all over the world.In China,lung cancer is the leading reason of all cancer in males,and occupies the second place in the female.At the same time,the mortality of lung cancer is the highest in all kind of cancers.According to WHO,there will be 2,500,000 people die of lung cancer until 2030.Most of the patients with lung cancer was at a late stage when they are diagnosed,which is the leading reason of high mortality.The only chance we can choose is systemic chemotherapy and limited targeted therapy and immunotherapy.However,in the case of systemic chemotherapy,the five year survival rate of lung cancer patients is still less than 10%.One of the reasons for this low survival rate is the difference in sensitivity to chemotherapy drugs.It is still a problem to be solved urgently in the treatment of lung cancer by the lack of reliable index before the chemotherapy and the guidance of the clinician to choose the best chemotherapeutic drug.Therefore,it is necessary to explore a method that is individualized and fully reflect the individual characteristics of patients and guide clinical chemotherapy.Primary cell culture is to obtain cells、tissues or organs directly from patients.Compared with the mature cell lines that commonly used in the study,the primary cultured cells can more accurately reflect the characteristics of the body and represent the biological and clinical characteristics of the patients.The primary culture of lung cancer is to obtain tissue samples by operation,and the primary culture is by enzyme digestion and tissue culture.However,most lung cancer patients are diagnosed with advanced lung cancer and lost the chance of surgical treatment.At the same time,when the cells of tissue culture were primary cultured,the growth environment of the cells changed greatly,and whether it would be selected two times in vitro was discussed.Therefore,the feasibility of tissue derived primary culture cells for the detection of chemosensitivity is still to be determined.This experiment attempts to lung cancer tumor cells were cultured in vitro,considering that most of the patients have been in advanced stage at diagnosis,lost the chance of operation,so the choice of lung cancer patients complicated with pleural effusion,pleural effusion for tumor cells were cultured and identified,and its detection sensitivity to chemotherapeutic drugs in vitro,the method is simple,safety,growth environment and more close to the specimen,is expected to provide a clinical basis for the next step of individualized treatment.ObjectiveWe tried to culture tumor cells from lung cancer in vitro,taking into account the majority of patients have been in advanced stage when they are diagnosed,losing the chance of surgery.So we choose the samples from lung cancer patients complicated with pleural effusion.Pleural effusion of lung cancer in isolated cells were cultured in vitro by immunofluorescence and model of nude mice to establish in vitro identification of success.MTS assay was used to test the identification of cell lines after the clinical commonly used chemotherapy drug sensitivity,compared with clinical data,to judge whether it has the prediction of tumor drug sensitivity and clinical treatment effect.MethodsWorking closely with the First Affiliated Hospital of Zhengzhou University,collecting samples that eventually diagnosed as pleural under aseptic conditions 100 mL pleural effusion in 250 mL centrifuge tube,brought to the laboratory within 30 minutes.After treatment,the DMEM/F12+GlutaMAX-l(1 x)medium containing bovine serum of was cultured in 5%CO2,5%O2 three,a culture box of.When the cells were passaged to the 20 generation,the cells had similar morphological characteristics and could be passaged stably.At the same time,we detected the cultured cells by immunofluorescence(Pan-CK,TTF-1,P53,Ki67)and subcutaneously transplanted tumor in nude mice.In vitro cell proliferation assay was performed to identify the tumor cells.At the same time using MTS colorimetric determination of the hair cells to chemotherapy drug cisplatin,carboplatin and pemetrexed and gemcitabine,vinorelbine,Changchun gefitinib sensitivity was determined,in contrast to its drug sensitivity consistent with clinical practice.ResultsOne lung adenocarcinoma cell lines were isolated and cultured successfully.The tumor cells were identified by immunofluorescence and subcutaneous transplantation in nude mice.The results showed that the sensitivity of the cell line was more sensitive to pemetrexed,platinum and gefitinib,and the sensitivity to gemcitabine and vinorelbine was less than that of the control group.This is consistent with the clinical treatment of the patient.ConclusionPleural effusion can be used as the primary cell culture specimen,which is easy to separate and purify.The results of drug sensitivity test of the lung cancer cells in vitro are consistent with the actual clinical chemotherapy sensitivity,which may be a new tool to predict drug sensitivity and guide individual drug use.