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The Study of Peripheral Blood Mononuclear Cell MHC Gene Expression in Early Acute Graft Rejection

Author: LiXiangDong
Tutor: ZouXiong
School: Shandong University
Course: Clinical Laboratory Science
Keywords: Host MHC Peripheral blood mononuclear cells (PBMC) Acute graft rejection (AGR) mRNA relative quantitative New Zealand white rabbits Pseudomonas aeruginosa cyclosprin Prednisone
CLC: R617
Type: PhD thesis
Year: 2007
Downloads: 328
Quote: 0
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Abstract


Organ and tissue transplantation is one of the most medical achievements in 20 century, now transplantation has became the most effective therapy measure when organ and tissue function failure. Allograft is familiar in clinic, no matter what type of transplantation, as long as donor express protein or molecule which different with recipient, the implant will be rejected. How diagnose acute graft rejection (AGR) in earlier stage is very important to clinical doctor.In recently,clinical doctor observe acute graft rejection mostly depend on the symptom that of patients,especially the function marker of graft organ,in spite of which of them, they detected AGR later,put off the cure time.Now a great deal of research about detecting graft rejection have been done . Peripheral blood lymphocyte CD4/CD8 ratio, cytokine like IL-2、IL-4、IL-10、TNF-α、and IFN-γ, and enzyme、protein 1、Complement、adhesion molecules and serum soluble CD30 (sCD30) levels also have relation with graft rejection, But they all have some defect in sensitivity, specificity and early AGR detecting.Up to now ,it haven’t find a generally recognized, noninvaded, good sensitivity and specificity marker that can used in clinical detecting, our objective in this study is to look for better AGR marker.Most of peripheral blood mononuclear cells (PBMC) were lymphocytes and monocyte, a majority of them were lymphocytes. MHC-I (major histocompatibility complex, MHC )almost on all nucles cells, the expression of MHC-I most popular on peripheral blood leukocyte, now it is certain that the target antigen reconiged by graft rejection is MHC molecule expressed on the surface of graft, MHC is very important in transplantation. What change of host MHC when acute graft rejection occurred post organ transplantation? Little research has done, because peripheral blood is easily to detect, we look as peripheral blood mononuclear cells MHC a representative of host MHC to study the change of host MHC, we hope to find better AGR marker.This exprement studied the changes of peripheral blood mononuclear cells MHC post New Zealand white rabbits skin graft, because both AGR and infection can result in MHC unusual, we also studied the changes of peripheral blood mononuclear cells MHC in infected New Zealand white rabbits. We select skin graft rabbits as animal model, one reason is rabbits are relatively big animal, their blood sample can be drawn continuous and have no effect on them.The other reason is AGR can be observed easily in skin graft compared with internal organ,so we selected New Zealand white rabbits as animal model.We imitate organ graft in human, New Zealand white rabbits skin autograft as experiment control, skin allograft as experiment group, to observe the changes of peripheral blood mononuclear cells MHC. We selected Pseudomonas aeruginosa as presentative in the study of New Zealand white rabbits skin infection, studied the changes of peripheral blood mononuclear cells MHC post injecting in Zealand white rabbits skin.In the study of peripheral blood mononuclear cells MHC gene expression in acute graft rejection, frifteen New Zealand white rabbits were randomly divided into three study groups: three rabbits in the autografted control group, six rabbits in the experiment group 1, the other six rabbits in the experiment group2. Experiment group 1 was given daily intramuscular doses of cyclosprin (CsA) (5mg/kg).When the skin graft was end, we observed the time of skin graft rejection, rabbits blood sample were drawn every other day and obtained peripheral blood mononuclear cells, detected the MHC I and MHC II gene mRNA expression by real time PCR,three days after skin graft,little skin sample were used as pathology detecting every two days. Skin grew well in autografted control group, the colour and hardness always normal, no AGR occurred. In Experiment group 1 and Experiment group2 scattered monocytes in derma two days before AGR, showed many lymphocytes and monocytes in derma when AGR occurred, no neutrophilic granulocyte infiltrated, it confirmed that AGR ocorred and no postoperation infection. The times for skin allograft rejection were as follows: CsA-treated allografted group (7.75±0.72)days, untreated allografted group (6.57±0.53) days.MHC I and MHC II gene mRNA levels did not show any obvious change in the autografted control. MHC I gene mRNA levels had a slow increase in the CsA-treated allografted group, but increased slowly and reached the highest when macroscopic appearance of graft rejection, no obvious change in the untreated allografted group. MHC II gene mRNA reached the highest level 2-3days before graft rejection appeared macroscopically in the CsA-treated allografted group and untreated allografted group, soon decreased to the low level.AGR occurs in a few days through two weeks after graft operation. Cell immunity plays an important role in this process that CD4 and CD8 T cells are activated. Most of PBMC are lymphocytes, and the majority of them are T- lymphocytes. One possible explanation for above MHCmRNA expression post skin graft was that when AGR occurred, lymphocytes were activated, cell immunity and humoral immunity enhanced, therefore, PBMC expressed more MHC gene mRNA, which resulted in more expression of MHC molecules.We also detected the change of blood routin and MHC gene mRNA expression. Eight New Zealand white rabbits were randomly divided into two study groups: three rabbits in the control group, five rabbits in the experiment group.The five rabbits in the experiment group subcutaneous injected Pseudomonas aeruginosa one day before experiment, the concentration of Pseudomonas aeruginosa was 5×10~9CFU.The three rabbits in the control group subcutaneous injected the same volume physiological saline. The experiment lasted eight days. Rabbits blood sample were drawn every two days, at first the blood routin were detected, then obtained peripheral blood mononuclear cells from remainder anticoagulative blood, detected the MHC I and MHC II gene mRNA expression by real time PCR.The result showed that the total nucles cells and neutrophilic granulocyte cells had no obvious change in the control group, the total nucles cells and neutrophilic granulocyte cells had obvious change in the experiment group. PBMC MHC I and MHC II gene expression had no obvious change in the control group, PBMC MHC I and MHC II gene expression had obvious change in the experiment group.It is easy to detecte bacteria infection, the total nucles cells and neutrophilic granulocyte cells in blood routin showed acute bacteria infection. When AGR ocorred, they had no obvious change in the control group and experiment group 1,2. So we think that when detecting PBMC MHC gene expression, at the same time detecting the blood routin. If no obvous increasing of total nucles cells and neutrophilic granulocyte cells and neutrophilic granulocyte cells ratio, but PBMC MHC gene expression increased, we can exclude the increasing of MHC gene expression caused by bacteria infection, so we can diagnosed AGR.Due to the majority of virus, especially cytomegalovirus(CMV) infection, PBMC MHC gene expression decreased, so if MHC gene expression increased, in the main can exclude virus infection, we can diagnosed AGR.Because MHC molecule had abundant polymorphism, it is difficut to obtain appropriate antibody. Real time RT-PCR is a rapid and hypersensitive experimental method, we knew that MHC molecule had abundant polymorphism, in the past it is diffcult to quantitate MHC molecule mRNA because MHC molecule had abundant polymorphism, we are the first study the conservative reigon of MHC, desigen the prime that span conservative reigon of MHC, so it is easly to quantitate MHC molecule mRNA, this make innovation in the world. In addition, PBMC can be easily obtained. The above results showed that PBMC MHC gene mRNA expression can be considered as an earlier no invasion marker to detect AGR, it had a obvious increase trend 2-3days before graft rejection appeared macroscopically, compared known marker it is earier, especially compared with used marker in clinic now, so we can use PBMC MHC gene mRNA expression as a early agr decting marker. But our experiment studied the chang of mRNA by housekeeping gene P-actin, it had no standarding in standar and method of quantitative, these are the shortcoming, we should look for better method of mRNA expression.Now it is certain that the target antigen recognized in graft rejection mostly are MHC molecule expressed on the surface of the graft. CsA suppress graft reject partly because CsA decreased MHC gene expression? Different study showed different result, some research showed CsA decrease MHC gene expression, but the research of Lim SW’s showed CsA increased MHC gene expression. We used New Zealand white rabbits in our experiment, to study the change of PBMC and organ ( liver, spleen, kidney and thyroid) post CsA intramuscular injection, the experiment lasted thrity days, to ddetect host MHC gene expression affected by CsA. It reported earier that glucocorticoid decreased MHC gene expression, but this time we detected systemly the change of PBMC and organ ( liver, spleen, kidney and thyroid) post taking prednisone.In the experiment of MHC gene expression affected by CsA, thriteen New Zealand white rabbits were randomly divided into three study groups: three rabbits in control group, five rabbits in low dose CsA treated group, five rabbits in high dose CsA treated group.The rabbits in low dose CsA treated group were given daily intramuscular doses of CsA (5mg/kg ), the rabbits in high dose CsA treated group were given daily intramuscular doses of CsA (20mg/kg ), the rabbits in control group were given daily intramuscular same volume physiological saline.The experiment lasted thrity days. Rabbits blood sample were drawn before experiment and every five days post experiment, detected blood serum glutamic-pyruvic transaminase(GPT), glutamic oxaloacetic transaminase(GOT), blood urea nitrogen(BUN) and creatinine(Cr) level, 1ml rabbits blood sample were drawn every five days post experiment, then obtained peripheral blood mononuclear cells, detected the MHC I and MHC II gene mRNA expression by real time PCR. When the experiment ended in day30, CsA -treated group and control group rabbits were killed by airebolism, their organ like liver, spleen, kidney and thyroid were drawn, detected the MHC I and MHC II gene mRNA expression by real time PCR.When the experiment ended in day30, New Zealand white rabbits in high dose CsA treated group glutamic-pyruvic transaminase(GPT): average value 35IU/L before experiment, average value 73IU/L when experiment was end ; glutamic oxaloacetic transaminase(GOT): average value 38IU/L before experiment, average value 78IU/L when experiment was end; urea nitrogen: average value 6.13mmol/L before experiment, average value 21.72 mmol/L when experiment was end ; creatinine level: average value 62μmol/L before experiment, average value 274μmol/L when experiment was end. The control group rabbits blood serum GPT, GOT, urea nitrogen and creatinine lever showed no change when the experiment was ended compared with the lever before experiment. When the experiment ended in day30, the pathology detection of liver and kidney in control group and low dose CsA group New Zealand white rabbits had no obvious change. The live showed acute CsA intoxication in high dose CsA group New Zealand white rabbits, including cholestatic in intrahepatic duct, the red blood cell increased in intrahepatic duct, and liver hepatic cell showed ballooning degeneration. The kidney showed acute CsA intoxication in high dose CsA group New Zealand white rabbits, including the red blood cell increased peritubal capillary and proximal convoluted tubule epithelial cell showed vacuolar degeneration.These result showed high dose CsA (20mg/kg ) had damaged the liver and kidney of experimental rabbits, CsA (20mg/kg) was indeed high dose CsA.The result of MHC gene mRNA expression showed that PBMC MHC I and MHC II gene mRNA expression increased significantly in day5-day10, there MHC I and MHC II gene mRNA expression decreased to the lever before experiment after day 15. When the experiment ended in day30, host organ (liver, spleen, kidney and thyroid) MHC I and MHC II gene mRNA expression had no obious change.Our study same as Lim SW’s showed CsA increased MHC gene expression. But CsA increase MHC gene expression for a time, only in day5-10, when continued to use CsA, MHC I and MHC II gene expression could not increase. Maybe host organ MHC I and MHC II gene mRNA expression also increased for a time treated with CsA, because only when the experiment ended we could detect host organ, there increasing trend had ended, so MHC gene expression showed no obvious change in experimental group compared with control group. CsA suppressed graft rejection because CsA binds to the cyclophilins (Cyps), it converge upon the calcium- and calmodulin-dependent serine-threonine protein phosphatase calcineurin (CaN). CsA complex can bind to calcineurin, thereby inhibiting its phosphatase activity. Our study showed that CsA can increase MHC gene expression for a time, it maybe CsA increased some cytokine expressed, this need more experiment to prove. This is the first time put forward that CsA can increase MHC gene expression for a time.It has a long history that glucocorticoid used as immunosuppressant post organ transplantation in clinic. Now it is thought that the action mechanism of glucocorticoid is that they suppressing activated immune cell cluster to graft organ in" recognition stage" and cleave the activated immune cell, so glucocorticoid can prevent immune cell from recognizing immune antigen in graft organ.Now it is certain that the target antigen recognized in graft rejection mostly are MHC molecule expressed on the surface of the graft. Acute graft reject occurs in a few days through two weeks after graft operation. Cell immunity plays an important role in this process that CD4 and CD8 T cells are activated. MHC molecule expressed on the surface of the graft can be recognized by CD4 and CD8 T cells through direct recognition or indirect recognition.We used New Zealand white rabbits in our experiment, used the method of real time PCR, to study the change of PBMC and organ MHC gene mRNA expression in New Zealand white rabbits treated with high dose prednisone. Eight New Zealand white rabbits were randomly divided into two study groups: three rabbits in control group, five rabbits in experiment group. The New Zealand white rabbits in experiment group were given daily doses of prednisone tablet (20mg/kg )dissolved by physiological saline, control group was given the same volume physiological saline, this experiments continued 8 days.1ml rabbits blood sample were drawn every in day4 and day8, then obtained peripheral blood mononuclear cells, detected the MHC I and MHC II gene mRNA expression by real time PCR. The result showed that PBMC MHC I mRNA expression decreased significantly in day4 and day8, PBMC MHC II mRNA expression showed obviously low in day 4, showed significantly low in day 8, Prednisone-treated group organ(liver, spleen, kidney and thyroid) MHC I and MHC II mRNA expression decreased obviously compare with control group. MHC gene mRNA expression of PBMC dropped significantly than that of host organ.The study of Giuliani C showed that glucocorticoid decreased MHC class I gene expression in rat thyroid cells. The early study showed that prednisone inhibited the expression of MHC II gene expression in mouse macrophage. Our study showed that prednisone dropped host organ MHC I and MHC II gene mRNA expression broadly, MHC gene mRNA expression of PBMC dropped significantly than that of host organ. So we believe that can use PBMC as a representative of host organ to study the change of host organ post using glucocorticoid prednisonePrednisone can decrease host organ MHC gene mRNA expression broadly, graft organ as a part of the host organism post organ transplantation, so glucocorticoid prednisone also decreased MHC gene mRNA expression on the surface of the graft organ. It looks as glucocorticoid prednisone drop the target antigen recognized in graft reject, i.e. MHC gene expression on the surface of the graft organ, so it can suppress graft rejection to some extent.

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