| PART 1 Detection of circulating tumor cells in gallbladder carcinoma and its clinical significanceBackground Gallbladder carcinoma is a devastating malignancy and is prone to invasion of adjacent organs and metastasis with poor prognosis.Currently,circulating circulating tumor cells(CTCs)in the peripheral blood is thought to be to an important factor related closely to transfer.Therefore,the detection of CTCs in the peripheral blood of patients with gallbladder cancer has important guiding significance for the evaluation of patients' condition,the choice of treatment plan and prognosis judgment.Objective The purpose of this study is to detect CTCs in peripheral blood of patients with gallbladder carcinoma and its clinical application.Methods The peripheral blood of patients with gallbladder carcinoma who underwent surgery in laparoscopic department from December 2014 to December 2016 was collected and the CTCs were detected.We collected middle elbow vein blood 5ml.CTCs were isolated and enriched using YZY-CTC-BIOPSY made by Wu Han biotech company as experimental platform.CTCs were identified by patented technology.Immunofluorescence staining of CK8 / CK18 / CK19 / CK133/ CD45 / DAPI were used to confirm CTCs.The clinical data were analyzed retrospectively,including clinicopathological features,treatment methods,follow-up data and so on.The relationships between CTCs and prognosis were statistically analyzed.Results A total of 80 cases of gallbladder lesions were isolated from the peripheral blood.63 cases were confirmed as gallbladder carcinoma and 50 cases were positive for CTCs.The positive rate was 79.4%(50/63).The number of CTCs was range 0-19 / 5ml,with median 6 / 5ml,with an average of 6.43 ± 0.65.17 cases were confirmed as benign lesions of the gallbladder,with CTC positive rate 0%(0/17).In 63 cases of gallbladder carcinoma,radical resection was performed in 43 cases with two cases of TNM stage II,27 cases of stage III and 34 cases of stage IV.Patients were followed up for a period of 1 to 26 months with 100% follow-up rate.The 0.5-,1-,2-year survival rates were 77.8%,49.5% and 36.9% respectively,with median survival time 11.9 months.There was a significant correlation between CTCs and TNM staging,hepatic invasion and CA199.CTCs are also an independent risk factor for multivariate analysis of prognosis of gallbladder carcinoma,and can be used to judge the effect of disease treatment and monitor the progress of the disease.The prognosis of patients with both CTCs and CTM positive was worse than that of CTCs alone,and the median survival time was 10.2 months vs 3.4 months.Conclusion Isolation and identification of CTCs in peripheral blood of gallbladder carcinoma were successfully performed by CTC-BIOPSY combined with immunofluorescence staining.CTCs are a important indicator for the diagnosis,monitoring disease progression and prognosis of gallbladder carcinoma,and have a great clinical value in gallbladder carcinoma.PART 2 Application of exosomes micro RNA sequencing in early diagnosis of gallbladder canceBackground Gallbladder cancer is the most common malignant tumor of the biliary system,the incidence increased rapidly in recent years.The incidence of gallbladder cancer is relatively high in China,which has become a serious threat to the lives of the masses.Due to the lack of effective early diagnosis strategy,most patients are diagnosed at advanced stage.The majority of patients had lost the chance of radical resection,and the 5 year survival rate was less than 5%.Gallbladder carcinoma is considered to be one of the most aggressive and lethal malignant tumors.Therefore,the early diagnosis and early treatment of gallbladder cancer are particularly important.It is of great clinical significance to search for diagnostic markers of early gallbladder carcinoma.Objective Searching for early diagnosis markers of gallbladder cancer and its clinical application.Methods The differences of micro RNAs in the exosomes of 10 patients with gallbladder carcinoma(Tis N0M0 or T1N0M0)and chronic cholecystitis were collected respectively and sequencing analyzed in order to find the specific markers of gallbladder carcinoma.Results Exosomes micro RNAs were successfully extracted from 20 patients,successfully building and sequencing a database of 12 cases.The success rate was 60%,including 6 cases of gallbladder carcinoma and 6 cases of chronic cholecystitis group.At the current read depth,615 micro RNAs in exosomes were detectable,among which 186 mi RNAs had statistically differential expression.Compared with chronic cholecystitis group,56 micro RNAs differential expression were up-regulation in gallbladder carcinoma,and 130 mi RNAs were down-regulation.We successfully performed differentially expressed micro RNAs for target gene prediction and GO,KEGG function analysis.Conclusion This study demonstrated that a wide variety of micro RNA species are embedded in the circulating exosomes.A tumor-related specific marker of gallbladder carcinoma can be screened based on the sequencing of exosomes micro RNAs.We discovered there were 56 micro RNAs up-regulation and 130 micro RNAs down-regulation in gallbladder carcinoma.We also performed target gene prediction and GO,KEGG function analysis to the most up-regulation significant difference.The method is noninvasive,has little damage to the patient and is convenient for application.It has a relatively broad potential application value for the early diagnosis of gallbladder carcinoma. |
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