| Objective:Malignant Pleural Mesothelioma(MPM)is a rare,highly malignant and invasive tumor with insidious onset,complicated diagnosis and treatment,and low survival rate;the global incidence is severe,and the relative incidence in Dayao County,Chuxiong Prefecture,Yunnan Province,my country More seriously,the asbestos use rate in this area is extremely high,and the mortality rate of patients is on the rise.Although the treatment of MPM has made more progress,the overall prognosis is still poor.This study mainly takes tissue samples from patients with malignant pleural mesothelioma who were diagnosed with malignant pleural mesothelioma in Dayao County,Chuxiong Prefecture,Yunnan Province as the main research objects,and explores a more suitable primary cell culture technology and related identification methods for MPM cells.They are all cultivating malignant pleural mesothelioma cell lines in Dayao County,Chuxiong Prefecture,Yunnan Province,and doing a good job in relevant identification work and analysis of biological characteristics,in order to provide future research on its carcinogenic mechanism and molecular studies,anti-tumor drug sensitivity detection,Mapping tumor gene maps and searching for relevant treatment target genes provide effective models and lay a theoretical foundation.Methods:Tissues were collected from Dayao County,Chuxiong Prefecture,Yunnan Province,who were diagnosed with malignant pleural mesothelioma in the Thoracic Surgery Department of the First Affiliated Hospital of Kunming Medical University and had no previous treatment history.6 cases of specimens(pathologically confirmed)were isolated,screened,tissue block cultured,cell transfected,primary cultured,subcultured,digested and purified to establish cell lines,and were tested by light microscopy and Wright-Giemsa staining Observe the morphological characteristics of cultured cells under a microscope;through the CCK-8(Cell Counting Kit-8)detection box: draw the growth curve of cultured cells(Growth Fraction,GF),calculate the doubling time of cultured cells(Cell Doubling Time,DT);the expression of some mesothelial and epithelial markers was detected by immunohistochemistry;the expression of related genes in cultured cells was detected by RT-PCR;the identification of cultured cells was further verified by Western blot analysis.The relatively stable growth of cultured cells was screened,and the tumorigenicity experiment of immunodeficiency mice was carried out;all experimental subjects were selected from specific pathogen-free(Specific Pathogen Free,SPF)nude mice,cultivated under sterile conditions to observe the growth of the cells after planting The tumor formation rate,symptoms,mortality and other data of nude mice within 30 days were dynamically detected for weight and diameter of transplanted tumors;for tumor tissues with good tumor formation,they were mounted and fixed,and stained with hematoxylin-eosin.Cell staining(Haematoxylin Eosine,HE)was used for morphological observation under a microscope and immunohistochemical detection of the expression of relevant mesothelial markers,and the primary cells,cultured cells and transplanted tumor cells were sent to the sequencing institution for further identification.Results:Through the above experiments,a case of cell adhesion was successfully observed in this experiment.After primary culture and subculture of the cells,the obtained cell morphology was consistent with the malignant proliferation of mesothelial cells.Compared with the primary cells,changes occurred.Not obvious;consistent with the morphology of tissue specimens,no mycoplasma contamination observed,waiting for STR results.Through the dynamic observation under the microscope and the detection of CCK8 proliferation during the cell culture process,the results showed that the cell growth was relatively stable and vigorous,and the logarithmic phase of growth was 2to 3 days.The doubling time is about 26.8h,32.3h,27.1h.The cultured cells are mainly polygonal cells,some of which are irregular spindle cells,and the cell arrangement is paving-like,with a little cytoplasm and nuclei showing the formation of vacuoles;the nucleoli are obvious,of different sizes,and mitotic figures can be seen,etc.Morphological biology Features consistent with malignant pleural mesothelioma.In the stage of subculture,the positive expression of mesothelial markers in the cultured cells was detected by immunohistochemistry.Calretinin had the highest expression,the highest overall sensitivity,and the highest staining change.The nuclear expression was obvious,and the overall nuclear expression was positive.The rate is 81.8%.CD5 and WT-1 also had high expression.CD5 was mostly expressed in the cytoplasm,showing a diffuse distribution reaction,and 70.0% showed staining changes.CD141 mostly showed focal membranous reaction,and the staining change was 62.0%.Epithelial markers TTF-1,Napsin A,and CEA were not expressed in cultured cells.The relevant RNA was extracted,and RT-PCR analysis showed that Ba P1,cdnk2 a,and NF2 expression decreased,among them,NF2 mutations were relatively rare.Western blot analysis was performed against cultured cells and the results obtained were consistent with RT-PCR results;There were significant differences in the expression levels of each gene and protein among the related test results.The cultured cells with stable growth were selected and transplanted on nude mice.The tumor was highly tumorigenic.Part of the tumor was taken and made into paraffin sections.The HE staining method and immunohistochemical detection were used to identify the morphological changes of malignant pleural mesothelioma cells.Biological characteristics,the cultured cells have tumorigenic characteristics.In the stage of nude mouse transplantation experiment,immunohistochemical detection showed that CK5,Calretinin,CD141,WT-1,EMA and other proteins were positively expressed,and Calretinin and WT-1 were strongly positively expressed in the nucleus;CK5 and EMA were expressed in the cytoplasm higher expression.Conclusion(s):This study screened 6 tissue samples from patients with malignant pleural mesothelioma who were born in Dayao County,Chuxiong Prefecture,Yunnan Province,and diagnosed with malignant pleural mesothelioma through surgical treatment(surgical method: thoracotomy and subpleural biopsy)in our hospital.A stable growth MPM cell line was cultivated by culturing and other methods,and the nature of cellular immune response and genetic characteristics were detected by morphological observation,immunohistochemical detection,Western blotting,RT-PCR,etc.,and part of the identification of the cultured cells was completed.As of the time of writing this article,the growth of the cultured cells is still good,and the cells are not contaminated by mycoplasma,which will provide powerful help for later related chromosome analysis,tumor microenvironment research,and target gene search.The established malignant pleural mesothelioma cell line,the cultured cells have good growth activity,the cultured cells were used to subcutaneously transplant nude mice,the tumors were successfully developed,made into paraffin sections,and the morphology and histopathological analysis were performed.The local growth and regional spread of the lesions were compared with clinical The incidence conditions are very similar,and the histopathological results suggest that the relevant mesothelial markers are expressed to varying degrees,consistent with the manifestations of mesothelioma,and similar to the immunohistochemical results of the original human tumor.Successfully completed the tumorigenic nude mouse planting experiment,laying the experimental foundation for further research on tumor carcinogenesis and mechanism;providing effective model resources for MPM anti-tumor drug screening. |
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