Study On Guiding The Surgical Resection Of Hepatobiliary Malignancy By The Fluorescence Navi Ation Technique Based On Fluorescent Tracer

ObjectiveHepatobiliary malignancies include liver tumors and biliary tract tumors,which are of high malignancy,difficult operation and poor prognosis.Radical resection is still the most effective method at present,but the high postoperative recurrence rate is always the difficulty for surgeons to study.Tumor resection integrity,tumor number,vascular invasion and lymph node metastasis are the main factors affecting long-term survival after surgery.In order to improve the prognosis and improve the surgical effect,surgeons have always been exploring the purpose.The development of molecular imaging has provided an innovative and effective method for improving surgical results.ICG fluorescence imaging technology can improve the RO resection rate of primary liver cancer and has the ability to find new cancerous lesions,thus potentially reducing the recurrence rate and improving the prognosis.However,more well-designed prospective clinical studies are still lacking to confirm the effect of this technique on the survival rate and recurrence rate of patients with liver cancer.Because this new technique has not been used in clinic for a long time,there is no corresponding standard for clinical application.Meanwhile,intraoperative ultrasound has been widely used in clinical practice,which can accurately locate tumors,identify tumor boundaries and anatomical relations with peripheral blood vessels and bile ducts.lt is able to find small lesions and tumor plugs that are difficult to be found in preoperative imaging examination,which can help make surgical plans and guide tumor resection.Some of its functions are the same as fluorescence navigation technology,so the value of the two in the same function is still lack of strict case-control studies to provide data basis.Failure of icg-specific imaging for cholangiocarcinoma and extrahepatic cholangiocarcinoma also limits its clinical application.In order to promote the standardization of the clinical application of this new technology and solve the above clinical problems,this study explored the effect of this technology on the prognosis of patients with primary liver cancer through a prospective study of ICG fluorescence-guided liver tumor resection.The method and value of the new method were compared with that of intraoperative ultrasound.At the same time,a new molecular probe cy7.5-rgd/MSNs was designed to specifically bind the bile duct carcinoma tissues,which is expected to apply the molecular imaging method in the operation of bile duct carcinoma and improve the surgical treatment effect of bile duct carcinoma.Methods(1)Choose our Hepatobiliary surgery specified time period of fitting the liver tumor resection of primary liver cancer patients with continuous 103 cases randomly divided into fluorescent navigation group and routine treatment,fluorescence navigation group using fluorescence navigation technology lead performer guided liver tumor resection,in comparison with conventional surgery group,the use of the conventional surgical method for liver tumor resection,two groups of patients followed up on January 1,2019 patients perioperative and postoperative relapse and death of the short-term and long-term.Then,through statistical analysis,the value of ICG fluorescence imaging technology in surgical treatment of patients with primary liver cancer was discussed.(2)Choose our Hepatobiliary surgery specified time period of fitting the liver tumor resection of primary liver cancer patients with continuous 50 cases,randomly divided into fluorescent navigation group and intraoperative ultrasound group,fluorescence navigation group using fluorescence navigation technology guide cancer resection of 25 cases,intraoperative ultrasound group completely using intraoperative ultrasound guided surgical removal of the 25 cases,record the complete resection rate of preoperative tumor known the ability of sexually transmitted diseases and found new carcinoma,have difference contrast data through statistical analysis,to explore the value of the two in resection of primary liver cancer surgery.(3)With cholangiocarcinoma tissue high expression of integrin ?V?6 as the target,and integrin ?V?6 specific binding peptides for theoretical support,and design a new type of fluorescent molecular probes Cy7.5-RGD,through exploring the probe for synthesizing method,the chemical characterization of molecular probe detection,cell toxicity experiment,targeting specific evaluation experiment in vitro,a tumor-burdened nude mice in vivo metabolic method and probe in the effect of body target experiment and discusses the basic research of the new molecular probes the indicators(4)Choose our Hepatobiliary surgery specified time fitting line of extrahepatic bile duct carcinoma in 37 patients with radical excision surgery,fresh specimens of bile duct carcinoma after resection for probe spraying,soaking,washing and fluorescence imaging technique is adopted to improve the imaging,records of tumor tissue and normal bile duct tissue imaging results,and to distinguish between tumor cases,tissue samples send pathologic examination,and according to the immunohistochemical results according to integrin alpha V beta,6 high and low or no expression is divided into two groups,statistical analysis and fluorescence imaging technique for tumor detection rate and detection of integrin ?V?6Results(1)(1)the results showed that in the fluorescence navigation group,the shortest distance between the tumor and the liver resection edge was 2 cases(4.0%,2/50)adjacent to the liver section(<0.1cm),and 8 cases(16.7%,8/48)in the conventional surgery group.The difference was statistically significant.Survival rates were significantly different at 3 months postoperatively compared between the two groups with and without adjacent incisional margins.?The 98 patients in the two groups were followed up to January 1,2019.There was no significant difference in the overall survival time(p=0.688>0.05),tumoral survival time(p=0.716>0.05)and overall recurrence rate(P=0.993>0.05)between the fluorescence navigation group and the conventional surgery group.lt indicates that fluorescence navigation technology has no significant effect on the overall recurrence rate and overall survival rate of primary liver malignancies.There were statistically significant differences in the 6month survival rate(p=0.039<0.05)and in the recurrence rate at 2,3,6 months after surgery(p=0.038,p=0.046,p=0.046)between the fluorescence navigation group and the conventional surgery group.This indicates that fluorescence navigation technology can reduce the short-term recurrence rate of primary liver cancer and improve the short-term survival rate.?comparison of postoperative recurrence rates between the two groups with tumor size between 3cm and 5cm:3 months(p=0.026<0.05),6 months(p=0.009<0.05),and 36 months(p=0.017<0.05)showed statistical difference.The survival rates of the two groups with tumor size between 3 cm and 5cm were statistically different at 24 months(p=0.026<0.05).The recurrence rate and survival rate of tumor<=3cm and tumor>5cm showed no statistical difference.(2)?fluorescence navigation technology conducted real-time guided resection of preoperative known lesions in 25 patients in the fluorescence navigation group,and no residual cancer cells were found in the pathological results of cut edges of isolated specimens after tumor resection.The pathological results of the incisional margin of 25 patients in the intraoperative ultrasound group showed that the incisional margin of 1 patient had residual cancer cells,and the complete resection rate(R0)of the tumor was 96%(24/25),and the residual rate(R1)under the microscope was 4%(1/25).? the shortest distance between the excision edge of the in vitro specimens and the tumor was more than 0.8cm,and there were 1 case(4%)with less than 1cm,and 24 cases(96%)with more than 1cm.The shortest distance between the intraoperative ultrasonic excision edge and the tumor was 0.1cm(0.1cm-3.2cm)on average.The shortest distance between the cancer tissue and the liver section was 0.1cm(1 case,4%),7 cases(28%)less than 1cm,and 18 cases(72%)greater than 1cm,which was statistically different from the fluorescence navigation group(p=0.049).?The sensitivity,specificity and false positive rate of fluorescence imaging were 71.4%,11.1%and 88.9%,respectively.The sensitivity,specificity and false positive rate were 42.9%,88.9%and 11.1%respectively.(3)?TEM and SEM were used to observe the Cy7.5-rgd/MSNs molecular probe,and the distribution of water and particle size was measured between 75-135nm,with an average of about 93nm.The absorbance of the targeted molecular probe at 808nm was detected by UV spectrophotometer UV-vis,and the light stability of the probe in serum was proved to be good.?The cell survival rate of the probe group was 98.12±1.55%?96.21±2.10%?95.60±1.30%?97.44± 1.87%?93.50±0.90%,and 97.10±2.10%,respectively.There was no significant difference in the cell survival rate between the probe group and the control group(P=0.132>0.05).QBC939-GFP cells were observed by near-infrared fluorescence microscopy,and probes were collected to generate fluorescence.?The results of fluorescence analysis of tumors and organs in vitro were consistent with the results of distribution metabolism experiment in vivo.(4)After the failure of the experiment in 14 cases,there were 23 cases of cholangiocarcinoma,and fluorescence could distinguish between tumor and normal tissue in 14 cases(60.9%).Immunohistochemical results showed that there were 16 cases(69.6%)with high expression of ?V?6 and 6 cases(30.4%)with low expression or no expression.Of the 16 patients with cholangiocarcinoma with high expression of ?V?,13(81.3%)could be distinguished by fluorescent probes,and the sensitivity and specificity of fluorescence in the diagnosis and screening of high expression of ?V?6 were 81.3%and 85.7%,respectivelyConclusions(1)According to the analysis results,the fluorescence navigation technology based on ICG can better ensure the safe distance of tumor resection when cutting off the deep liver parenchyma.Adjacent to the liver resection margin is an important risk factor for short-term survival after hepatectomy.Fluorescence navigation technology can reduce the short-term recurrence rate of primary liver cancer and improve the short-term survival rate.This technique is valuable for recurrence and 2-year survival in patients with primary liver caneer whose tumor size ranges from 3cm to 5cm.This technique ean improve the surgical effect of primary liver cancer,and ultimately reduce the short-term recurrence rate of primary liver cancer patients and improve the short-term survival rate.(2)This indicated that the fluorescence navigation technology could better ensure the safe distance between the tumor and the liver resection margin and improve the R0 resection rate of the tumor.In addition,fluorescence navigation technology can be used as a supplementary resection edge detection for intraoperative ultrasound resection of tumors,and the final pathological results showed that all the 25 patients in the intraoperative ultrasound group achieved the effect of RO resection.Fluorescence navigation technology has a higher sensitivity than intraoperative ultrasound in the detection of new cancer lesions,and is more comprehensive in the detection of cancer lesions,but a higher false positive rate is still the direction to be avoided in further research.Combined fluorescence navigation and intraoperative ultrasonography were used to detect the remaining liver,which can make up for each other and achieve a higher detection rate of cancer lesions.(3)We successfully synthesized and constructed a fluorescent nanoprobe Cy7.5-RGD/MSNs targeting the integrin ?V?6 receptor of cholanglocarcinoma.The MSNs loaded with silica spheres showed high specificity for ?V?6 receptors,good biocompatibility,light stability,high water dispersion and high targeting in tumor-bearing nude mice.(4)Through the analysis of results and the exploration of clinical application methods,the novel molecular probe Cy7.5-RGD/MSNs can specifically target fresh tissue of cholangiocarcinoma,while the normal bile duct is not visible,thus achieving the purpose of distinguishing cholangiocarcmoma from normal bile duct.Especially in the bile duct carcinoma tissues with high expression of integrin aVp6 receptor,this molecular probe showed high specificity,which is expected to be a new probe and new technique to guide the fluorescence imaging of intraoperative bile duct carcinoma tissues.