Study On Microstructure,composition Distribution And Stone Forming Mechanism Of Gallstones

Purpose:By synchrotron radiation X-ray phase contrast micro tomography(SR μCT)study the ultrastructure of gallstones,analyze the distribution of components corresponding to threedimensional ultrastructure,so as to scientifically classify gallstones,classify different ultrastructure and component distribution,and look for possible stone forming differences.Using Fourier transform infrared spectroscopy(FT-IR)to qualitatively analyze the component distribution of gallstone in three-dimensional spatial structure can further clarify the difference of component distribution of gallstone in spatial structure.According to these differences,the differences of serum bile acids(BAs)among patients with different classification of gallstones were studied,and the possible mechanism of gallstone formation was found.Methods:(1)Acquisition of human gallstone specimens and statistics of clinical baseline dataA total of 100 gallstone specimens from patients with gallstones were included in this study.We retrospectively selected patients who met the following criteria from the existing database(inclusion criteria:they met the surgical criteria for gallstones,and the gallstones were confirmed by routine B-ultrasound before operation).Exclusion criteria:tumor,hypertension,diabetes mellitus,renal insufficiency,acute or chronic liver disease,cardiovascular or cerebrovascular diseases,or chronic oral administration of various drugs.Before operation,it was in the onset of acute cholecystitis(within 1-3 weeks)and liver dysfunction:such as jaundice,common bile duct stones,biliary pancreatitis and other diseases that may affect liver function).All participants were hospitalized patients.They were carefully asked about their medical history,physical examination and routine biochemical examination.Samples were collected from each enrolled patient 200μl serum was stored in-80℃ refrigerator for determination of bile acid family.Relevant data were recorded and the clinical baseline data were counted.Since the operations were completed under laparoscopy,after obtaining the gallstones,they were quickly washed with normal saline and dried naturally,stored in the sealed specimen belt,and prepared to start the experiment within 2 weeks.(2)Synchrotron radiation micro CT(SR μCT)using Shanghai light source BL13w1 line station,complete 100 cases of gallstones 3-9μm thin-layer CT image nondestructive acquisition,and analyze the effectiveness of the collected data.(3)The three-dimensional structure of thin-layer CT image data was reconstructed with Amira 5.4.3 software(visage,Berlin,Germany),and the three-dimensional spatial structure and component distribution of gallstone were obtained.(4)The spatial structure and composition distribution of gallstones were classified,analyzed and looked for similarities and differences.(5)100 gallstones from the three groups obtained in the previous study were cut in half with a sharp art knife.The three parts of the core,perinuclear and nuclear surface of each included stone were taken with an ordinary optical microscope,and each part was marked and recorded.(6)The samples were analyzed by pressing method and Fourier transform infrared spectrometer(Nicolet 6700,USA)400-4000cm-1.Each sample was scanned 64 times and the average spectrum was obtained in absorbance mode.(7)By comparing the infrared absorption peaks of gallstones in each group with the values reported in classical literature,the infrared absorption peaks and the width range of dense peak sections in each group were collected,and their common or different characteristics were compared.(8)The characteristics of infrared spectra of gallstones are analyzed and summarized in combination with the three-dimensional microstructure and component distribution of gallstones,so as to find the relevant internal laws and possible differences.(9)Bile acid molecular detection:inclusion of subjects:the serum of the healthy control group was obtained.According to the previous research results and classification,half of the patients with cholesterol stones,mixed stones and bile pigment stones were selected from the gallstone group.The obtained serum was used to obtain laboratory data by ultra high performance liquid chromatography triple quadrupole mass spectrometry(uplc-tqms).(10)The experimental results of bile acid molecules were combined with the targeted metabolomics method of univariate and multivariate statistical analysis to study the serum BAs spectrum of healthy controls and patients with different types of gallstones,and the possible differential molecules of bile acids were counted.(11)Combined with the differences of three-dimensional micro structure and component distribution of gallstones,compare the differences between serum bile acid families in patients with different types of stones,and further study the effects of different bile acid molecules on the micro structure and component distribution of gallstones,It is speculated that there may be a stone forming mechanism.Result:(1)SR μCT showed that different types of gallstones showed three-dimensional structure with certain regular characteristics according to the content and distribution of cholesterol and bile pigment calcium.(2)Cholesterol stones and mixed stones have a periodic layered structure with cholesterol as the core and cholesterol as the periphery.The difference is that cholesterol,the main component of cholesterol stones,is relatively evenly distributed in gallstones,and the periodic layered structure is relatively sparse;The periodic layered structure of mixed stones from inside to outside,the gap between adjacent layers decreased significantly and the density increased significantly,indicating that the stone forming mechanism of the two may be different.(3)There is no obvious regularity in the internal structure of bile pigment stones,and calcium bilirubin is relatively evenly distributed in bile pigment stones.The surface structure of non infectious bile pigment stones is rough and disordered,while the surface structure of infectious bile pigment stones is similar to the coral reef formed by the continuous death and accumulation of coral insects,which may be the factor promoting the formation of stones.(4)Fourier transform infrared spectroscopy further confirmed the scientificity of micro CT in the study of gallstones.Cholesterol stone is a layered structure with cholesterol as the core,which is mainly composed of cholesterol.The core main component of mixed stones is the layered structure of cholesterol,the layered structure is obvious from inside to outside,the gap is obviously reduced,the characteristic absorption peak of infrared spectrum,and the characteristic absorption peaks of cholesterol,calcium bilirubin and calcium carbonate are mainly around the core.Bile pigment stones show a disordered structure with the main component of calcium bilirubin.In addition to the main components,gallstones contain esters,proteins and other substances,which are involved in the formation of gallstones.(5)The microstructure and composition distribution of gallstones in three-dimensional space can speculate and explain the common phenomena in clinic.Gallstones are loose and easy to be clastic or muddy,which are usually written as silt like stones in clinic.As the more close to the peripheral periodic layered structure,the higher the density and the higher the content of bile pigment and calcium carbonate,the common clinical stones are hard and smooth.Due to the large and uniform distribution of cholesterol,the texture of cholesterol stones is between the two,and the surface is rough.(6)There were overall differences in serum bile acids between the healthy control group and the cholelithiasis group:through statistical analysis,the plasma concentration of total secondary BAs in the cholelithiasis group was significantly higher than that in the healthy control group(P<0.001),while the ratio of total primary BAs was significantly lower(P<0.05)(7)There were significant differences in the abundance and of serum BAs between male and female patients in cholelithiasis group:the concentration of total primary bile acids in men was higher than that in women,and the concentration of total secondary bile acids in women was higher than that in men.The total primary bile acid concentration of the former was lower than that of the latter,and the total secondary bile acid concentration of the former was higher than that of the latter.The statistical analysis showed that P<0.05(P=0.041).(8)There was significant difference in the abundance of serum total primary bile acids and total secondary bile acids among patients with cholesterol stones,pigment stones and mixed stones(P<0.01).The concentration of serum total secondary bile acids in patients with cholesterol stones was significantly higher than that in patients with pigment stones and mixed stones,while the concentration of serum total primary bile acids was lower than the latter two.(9)GCDCA、GHCA、GUDCA、NorCA、THCA、THDCA and TUDCA were found in the serum of patients with cholesterol stones and mixed stones.The serum bas of patients with cholesterol calculi and mixed calculi were ranked by P value GHCA<GUDCA<GCDCA<THCA<THDCA<TUDCA<NorCA<0.05<TωMCA<TaMCA.It is mainly composed of different hydrophilic bile acids and mouse bile acids.(10)The level of hydrophilic bile acids with protective effect in serum in cholesterol stones was significantly lower than that in mixed stones,and the level of mouse cholic acid was also lower than that in mixed stones.The analysis of the functions of hydrophilic bile acids and mouse cholic acid involved in metabolism in vivo confirmed the differences in perinuclear microstructure and component distribution mechanism between cholesterol stones and mixed stones.Combined with the clinical experience in the treatment of cholesterol stones,the classic method:using the representative UDCA of hydrophilic bile acids to treat cholesterol stones has achieved certain curative effect,and the effect is poor in the treatment of mixed stones.Conclusion:1.Cholesterol is the main component around the core of cholesterol stone,and the layered structure is loose;The mixed stones are mainly composed of bile pigment calcium,calcium carbonate and cholesterol around the nucleus,and the layered structure is dense;Bile pigment stone is mainly composed of bile pigment calcium,which is loose and disorderly distributed.Therefore,from the microstructure and composition distribution of gallstones,it is explained that increasing the capacity and concentration of hydrophilic bile acid pool through UDCA treatment can dissolve cholesterol stones,and the increase of the total amount of bile acid pool is conducive to expelling the fragments of easily disintegrating bile pigment stones out of the biliary system,while the treatment of mixed gallstones is ineffective.2.The increase of serum total secondary bile acid level and the decrease of primary bile acid level are the main causes of gallstone susceptibility.The etiology of cholelithiasis changes the quality and quantity of total bile acid pool.The significant reduction of hydrophilic bile acid level is the main reason for the difference in the mechanism of cholelithiasis between cholesterol stones and mixed stones.The increase of serum cholic acid level reduces the metabolic synthesis of clear water bile acid,which is conducive to the formation of gallstones.3.GCDCA、GHCA、GUDCA、NorCA、THCA、THDCA、TUDCA and MCA can be used as new molecular targets for further study of the etiology of gallstones.