Morphology Of Pathologic Membrane In Inferior Vena Cava And Screening For Its Correlate Gene In Budd-Chiari Syndrome

The cause and development process of Budd Chiari syndrome (B-CS) is not clear until to now. Company all kinds of investigations in different angles, it emerges lots of theories for the disease cause, such as "growth malformation, thrombus formation, physico-damage and infection". Followed with depth-investigation, it was fond that there were many complicated procedure for genesis and development for B-CS gradually. Now, it is general consider that B-CS is correlate to living-region. Most B-CS patients in western countries are followed with some definite reasons such as abnormal coagulation or other reasons. While most of them in our country are protopathic, maybe relate to those reasons include sex, age, environment, labour intensity, life condition, sanitation, liver function and gene mutation. Because it is difficult to abtain the abnormal membrane from B-CS patients, most investigations about the reason for B-CS are limited in abnormal coagulation and individual gene mutation whether domestic or overseas. Contrarily, we still know few about the abnormal membrane for its origin, formation and development.This investigation is aimed to do some research for pathogenesis from all these aspect include morphology, cytokine, apoptosis and gene profile in B-CS, accordingly to establish a molecular biology fundament for advanced study in B-CS. Firstly, we observe the general characteristics of abnormal membrane in inferior vena cava (IVC). Secondly, we contrast and find out the difference from abnormal membrane, scar, and normal vessel wall of IVC through light microscope and electronic microscope. Thirdly, we try to find out the immunohistochemistry change in the tissue of abnormal membrane, and describing the expression of fibroblast growth factor receptor (FGFR), proliferating cell nuclear antigen (PCNA) and apoptotic cell (AC). Finally, we try to figure out the difference between the gene profiles of abnormal membrane and the normal vessel wall of IVC through gene-chips.Part OneInvestigation of Morphology for Pathologic Membrane in IVC MethodsEleven cases with abnormal membrane in IVC had been taken through radical surgery with Budd Chiari syndrome from October 2007 to December 2009 in the First Affiliated Hospital of Zhengzhou University. Meanwhile,10 cases with scar tissue and 12 with normal vessel wall tissue from IVC were enrolled into this investigation. We observed the characteristic of abnormal membrane in IVC and tried to find out the differences of tissue from abnormal membrane, scar, and normal vessel wall of IVC through light microscope and electronic microscope, followed with some immunohistochemistry examination aimed to describe the chemical change in the tissue of abnormal membrane, and describe the expression of FGFR,PCNA and AC.Result1 General view of abnormal membrane1.1 Through radical surgery we observed that abnormal membrane located in the distance not exceeding 3.0cm where IVC flow into right atrium,6 cases located in the region of 2.0cm far from right atrium. It looked white, thickness on borderline, and thinness on central, surface stretch to intima of IVC. Eight cases were horizontal and 3 cases were oblique. In 11 cases of B-CS,9 cases of membrane were easy to be separated from vessel wall. Vessel wall looked smooth while membrane exsected. Two cases were difficult to separated. Vessel wall looked rough after been sharply exsected. There were 9 cases with thrombus adhered to IVC and 5 cases with calcification point on membrane. It looked white and smooth for the exsected membrane after washed with water. The thickness of the membranes on the borderline was from 5.0mm to 9.0mm, the mean was 6.8±1.5mm. It was from 3.0mm to 6.0mm in the central, the mean was 4.6±1.0mm.1.2 Scar tissue was hard and outstanding from skin with grey color and translucence. It could be confirmed that the scar tissue infiltrated to all layer of skin.1.3 Normal tunica intima of IVC appeared white, Eustachian valve was found in 1 case of all 12 cases.2 Tissue Morphology Observation under Llight Microscope.2.1 Pathological membranes were composed of rough and chaotic collagen fiber and fibroblast with few capillary, most of which blocked. Edge of membranes are full of proliferative fibrous connective tissue and smooth muscle cells, some were Hyaloid degeneration. Nine cases of membranes accompany with blood vessel hyperplasia and thrombus adhere to vessel,8 cases with hyalinization,5 cases with tissue calcification and 7 cases with inflammatory cells infiltration, of which most were lymphocytes and few were plasma cells and eosinophilic granulocytes. Granulation tissue and new vessels could be found in some cases. dermis2.2 In scar tissue, few fibroblast and big amount collagen fiber could be found. Collagen fiber showed parallel or cross ways. They were melt with each other, and formed as rough collagen lacertus. Small blood vessel distributed rarely with inflammatory cells infiltration. Neither hair follicle nor eccrine sweat glands were found in scar tissue.2.3 Normal IVC tissue were composed of intima, tunica media and adventitia, which over jet with a layer of endothelial cell. Endothelial cells of IVC were oblate and well-arranged and adhered close with each other with an oval nucleus. The layer of smooth muscle under the endothelium is thicker. Cells in it most are Fusi form shape where there are few intercellular substance like collagen fiber and elastic fibers between each other. It is not very clear for the boundary between tunica media and adventitia.3 Expression of FGFR, PCNA and Cell Apoptosis Condition3.1 There were positive cells for expressing FGFR, PCNA in all disease membrane, scar tissue and normal IVC tissue. The positive rate for expression of FGFR in three tissue are separate 17.30±1.5,18.50±1.64 and 8.80±1.24, which for PCNA are separate 40.76±3.67,38.46±2.33 and 22.10±2.01. There are no significant difference (P>0.05) between pathological membrane and scar tissue for FGFR and PCNA. The expression of both FGFR and PCNA in pathological membrane are higher than normal IVC tissue (P<0.05).3.2 There were no difference (P>0.05) between pathological membrane and scar tissue for count of apoptosis cells. But the number of apoptosis cells in both of them was fewer than in normal IVC tissue.4 Electronic Microscope View4.1 There were large quantity of fibrocytes in pathological membranes, with few endothelial cells in the surface which were thin and flat with irregular muclear Most mitochondria in the endothelial cell took on such a phenomenon that crista and membrane appeared fusion together, which was blur or absence. Rough endoplasmic reticulum in endothelial cell swelled slightly, particle fusion or absence could be found. Parts of intima in pathological membrane was cracked or fallen, bounding towards lumens. ACs could be found occasionally but with decrescent volume.4.2 There were many collagen fiber arranged chaotically around the fibroblast, which took on fusiform shape with rough and big outstanding with big and ellipse nuclear which had clear nucleolus. Endoplasmic reticulum could be found in cytolymph of fibroblast with abundance of Palade's granule and mitochondria which were ellipse or round shape. There were much secretory vesicle adhere to the cell membrane. 4.3 The tunica intima in the normal IVC tissue were smooth with thin, flat and round endothelial cell overjet. Endothelial cells arranged closely and regularly like a strap parallel with the major axis of the vessel. Vascular endothelial cells were pavement epithelium cells, most of them without nuclear. Mitochondria, rough endoplasmic reticulum and pinocytosis vesicle could be found in endothelial cell, with few collagen fibers and elastic fibers arranged raritasly. Smooth muscle layers under the endothelium were very thick. Smooth muscle arranged regularly with Fusiform shape nucleus, few rough endoplasmic reticulum and mitochondria, there were also much myofilament around the nucleus. Collagen fiber, fat cell and blood capillary could be found in the adventitia.Part twoRelated Gene Screening for Pathologic Membrane of IVC in B-CSMethodsThree cases of tissues from pathologic membrane of IVC were collected and refrigerated. At equal pace,3 cases of normal tissues were also collected as control from normal IVC at the point close to right atrium. Total RNA of all samples had been extracted and qualified. Total RNA was transcribed into cDNA and labeled with Agilent fluorescence and was taken into Northern blot hybridization by gene-chips. After hybridization, chips werre scanned by laser scanner. All data were collected and analyzed automatically. Genes with different expression between pathologic membrane and normal tissue from control would be found out by Student T test (P<0.05 and FC>10 as significant deviation). According this, potential target gene profile was forecasted through special software.ResultThere were 9371 genes with different expression between pathologic membrane and control. Among them,4986 genes in pathologic membrane expressed up-regulated, while 4385 genes down-regulated. Some reported genes caused pathologic membrane formation, such as FVL,JAK2-V617F and G20210A were found in our data.Conclusion1 Fiber hyperplasia take the main part in the formation process of pathologic membrane in B-CS patients, as well as injury and inflammatory reaction.2 Gene expression level of FGFR,PCNA and AC may take part in the process of pathologic membrane formation, their expression level may relate to the progress and result of the pathologic membrane.3 Gene array can efficiently analyze the data and get the disease related gene profile, which can speculate some possible target genes. The different expression of these genes may participate in the process of pathologic membrane formation.