本文選題：肥大細(xì)胞 + 色甘酸鈉　； 參考：《上海交通大學(xué)》2014年博士論文

【摘要】：近年來，肥大細(xì)胞在移植免疫中的作用越來越被人們所重視。肥大細(xì)胞在其中的功能仍然有很多問題需要解決，如肥大細(xì)胞脫顆粒在肝臟缺血再灌注損傷中的作用、調(diào)控肥大細(xì)胞獲得的供肝前置狀態(tài)對(duì)受體生存的影響和移植后受體肥大細(xì)胞脫顆粒在急性排斥反應(yīng)中的作用等。 本研究通過大鼠肝臟缺血再灌注損傷模型、大鼠原位肝移植模型、大鼠異位心臟移植模型三個(gè)移植研究中的經(jīng)典模型，分別探討了以上三個(gè)問題。為比較全面地研究肥大細(xì)胞在器官移植免疫中的作用，最終改善移植預(yù)后提供理論依據(jù)。 第一部分肥大細(xì)胞脫顆粒對(duì)肝臟缺血再灌注損傷作用的研究 研究背景：肥大細(xì)胞脫顆粒參與了許多器官的缺血再灌注損傷。但是在最近的一篇研究中卻發(fā)現(xiàn)肥大細(xì)胞不參與大鼠肝臟的缺血再灌注損傷。我們對(duì)此結(jié)果提出異議。 材料和方法：我們使用大鼠在體熱缺血1小時(shí)然后灌注24小時(shí)的模型觀察肥大細(xì)胞在肝臟缺血再灌注損傷中的作用。用了兩種方法研究肥大細(xì)胞脫顆粒所產(chǎn)生的效應(yīng)。第一種是用肥大細(xì)胞膜穩(wěn)定劑色甘酸鈉穩(wěn)定肝臟肥大細(xì)胞；另外一種是用肥大細(xì)胞脫顆粒劑化合物48/80“耗竭”肝臟肥大細(xì)胞。使用肝臟甲苯胺藍(lán)組織學(xué)染色和ELISA檢測(cè)血清中肥大細(xì)胞特異性蛋白肥大細(xì)胞胰蛋白酶的水平對(duì)兩種調(diào)控方法的效果進(jìn)行評(píng)估。使用肝臟組織HE染色下的Suzuki評(píng)分、血清中的谷丙轉(zhuǎn)氨酶（ALT）和谷草轉(zhuǎn)氨酶（AST）、肝臟組織裂解液丙二醛（MDA）等指標(biāo)對(duì)肝臟損傷嚴(yán)重程度進(jìn)行評(píng)估。 結(jié)果：第一，肥大細(xì)胞脫顆粒在再灌注2h時(shí)達(dá)到高峰，而ALT和AST反應(yīng)的肝臟損害在再灌注6h后達(dá)到高峰；第二，之前“耗竭”皮膚肥大細(xì)胞的方法，也能達(dá)到所謂的“耗竭”肝臟的肥大細(xì)胞；第三，通過色甘酸鈉穩(wěn)定肝臟肥大細(xì)胞和化合物48/80耗竭肝臟肥大細(xì)胞的方法能夠減輕Suzuki評(píng)分、血清中的ALT和AST、肝臟組織裂解液MDA等反映的肝臟缺血再灌注損傷程度。 結(jié)論：肥大細(xì)胞脫顆粒促進(jìn)了大鼠肝臟的缺血再灌注損傷。 第二部分調(diào)控供肝肥大細(xì)胞獲得的前置狀態(tài)對(duì)大鼠原位肝移植后受體生存的影響及其機(jī)制研究 研究背景：移植后的免疫抑制治療會(huì)導(dǎo)致機(jī)會(huì)菌的感染和腫瘤的生成等副反應(yīng)。我們前面的研究表明，通過色甘酸鈉穩(wěn)定肥大細(xì)胞和化合物48/80“耗竭”肥大細(xì)胞后能減輕肝臟的缺血再灌注損傷。本部分旨在探討這兩種調(diào)控肥大細(xì)胞的方法處理供肝獲得的前置狀態(tài)對(duì)受體生存的影響及其機(jī)制。 材料和方法：用色甘酸鈉穩(wěn)定供肝肥大細(xì)胞（CRM組）和化合物48/80（CMP48/80組）耗竭供肝肥大細(xì)胞的方法預(yù)處理供肝，，獲得相應(yīng)的前置狀態(tài)。然后用雄性DA大鼠作為供體，雌性Lewis大鼠作為受體建立大鼠原位肝移植模型。用PBS預(yù)處理供肝作為陽性對(duì)照（PBS組）。雄性Lewis作為供體，雌性Lewis作為受體的同基因移植作為陰性對(duì)（Syn組）。 結(jié)果：PBS組的受鼠快速發(fā)生急性排斥反應(yīng)并在7.40±1.14天死亡。與PBS組相比，CRM和CMP48/80組的生存期明顯延長，分別為17.40±1.67和14.20±2.28天，排斥活性指數(shù)和肝功能也明顯改善(p 0.05)。 Th1的代表細(xì)胞因子IFN-γ在肝臟組織中的mRNA表達(dá)和血清中的蛋白水平均明顯降低；而Th2代表細(xì)胞因子IL-10在肝臟組織中的mRNA表達(dá)和血清中的蛋白水平均明顯升高。 結(jié)論：通過色甘酸鈉穩(wěn)定供肝肥大細(xì)胞和化合物48/80耗竭供肝肥大細(xì)胞獲得的前置狀態(tài)能夠減輕急性排斥反應(yīng)和延長移植物生存。這結(jié)果可能是通過使Th1/Th2免疫平衡向Th2方向傾斜達(dá)到的。 第三部分移植后調(diào)控受體的肥大細(xì)胞對(duì)急性排斥反應(yīng)的影響及其機(jī)制研究 研究背景：受體體內(nèi)肥大細(xì)胞在急性移植排斥中的作用還不十分清楚。本部分研究旨在探討移植后受體肥大細(xì)胞脫顆粒與否及其對(duì)急性排斥反應(yīng)的影響。 材料和方法：在體外實(shí)驗(yàn)部分中，我們分離獲得了大鼠原代皮膚肥大細(xì)胞（RSMC）和腹腔肥大細(xì)胞（RPMC）。然后分別檢測(cè)肥大細(xì)胞表面受體C5a的表達(dá)情況及其在C5a作用下能否發(fā)生脫顆粒；在體內(nèi)實(shí)驗(yàn)中，我們建立DA大鼠到Lewis大鼠的異位心臟移植模型，按照受體處理方式設(shè)計(jì)分組：注射PBS的PBS組、注射色甘酸鈉的CRM組、注射3mg/kg體重CsA的CsA3組、聯(lián)合注射3mg/kg體重CsA和色甘酸鈉的CsA3+CRM組、注射5mg/kg體重的CsA5組、聯(lián)合注射5mg/kg體重CsA和色甘酸鈉的CsA5+CRM組以及同基因移植組。觀察各組的生存期。分別在術(shù)后第5、10、15天取移植受體靜脈血進(jìn)行C5a、IL-10和IFN-γ的檢測(cè)；肥大細(xì)胞脫顆粒試劑盒檢測(cè)肥大細(xì)胞脫顆粒水平；取移植心臟進(jìn)行HE染色，觀察急性排斥情況；移植心臟組織提取mRNA，檢測(cè)IL-10和IFN-γ的轉(zhuǎn)錄水平。 結(jié)果：在體外研究中，58.69%~67.64%的RSMC表達(dá)C5a受體，僅1.23%~1.89%的RPMC表達(dá)C5a受體。經(jīng)過脂多糖（LPS）刺激的RPMC表達(dá)C5a受體的細(xì)胞占73.52%~81.14%。C5a可以引起RSMC和經(jīng)過LPS刺激的RPMC脫顆粒，但不能引起不經(jīng)刺激的RPMC脫顆粒。C5a誘發(fā)的肥大細(xì)胞脫顆�？梢员环蚀蠹�(xì)胞膜穩(wěn)定劑色甘酸鈉所抑制。在體內(nèi)實(shí)驗(yàn)中，大鼠異位心臟移植后的受體C5a和肥大細(xì)胞脫顆粒水平較同基因移植組高，而且排斥越重C5a和肥大細(xì)胞脫顆粒水平也越高。色甘酸鈉能降低受體肥大細(xì)胞脫顆粒水平。但是，CRM組和PBS組比、CsA3+CRM組和CsA3組比、CsA5+CRM組和CsA5組比，生存期均不能顯著延長。心臟排斥評(píng)分、心臟組織IL-10、IFN-γ的mRNA和血清中的IL-10、IFN-γ之間的水平也均無顯著差異。 結(jié)論：在異位心臟移植的大鼠體內(nèi)的確有C5a和肥大細(xì)胞脫顆粒水平的上升；C5a也能促進(jìn)肥大細(xì)胞脫顆粒；色甘酸鈉能夠降低肥大細(xì)胞脫顆粒程度，但最終無法減輕急性排斥反應(yīng)和延長移植物生存。
[Abstract]:In recent years , the role of mast cells in transplantation immunity has been paid more and more attention . The function of mast cells still has many problems to be solved , such as the role of mast cell degranulation in liver ischemia - reperfusion injury , the effect of regulatory mast cells on the survival of receptors and the role of receptor mast cell degranulation in acute rejection .

In this study , the model of rat liver ischemia - reperfusion injury , orthotopic liver transplantation model and ectopic heart transplantation model in rats were studied . The three problems were discussed . In order to study the role of mast cells in organ transplantation immunity , the theoretical basis for improving the prognosis of transplantation was provided .

Effect of first partial mast cell degranulation on ischemia - reperfusion injury in liver

Background : Mast cell degranulation was involved in the ischemia / reperfusion injury in many organs . However , in a recent study , it was found that mast cells did not participate in the ischemia / reperfusion injury in the liver of rats . We disputed the outcome .

Materials and Methods : The effects of mast cells on ischemia - reperfusion injury of liver were observed in rats after 1 hour ischemia and reperfusion for 24 hours . Two methods were used to study the effect of mast cell degranulation .
In addition , the liver mast cells were depleted by using a mast cell degranulation compound 48 / 80 . The effects of two methods were evaluated by histological stain and ELISA in serum . The severity of liver injury was assessed by using the Suzuki score under HE staining of liver tissue , alanine aminotransferase ( ALT ) and aspartate aminotransferase ( AST ) in serum , and malondialdehyde ( MDA ) in liver tissue .

Results : The first , mast cell degranulation peaked at 2 h reperfusion , while the liver injury of ALT and AST reaction reached the peak after 6 h reperfusion .
Secondly , the method of " depletion " of skin mast cells can also achieve the so - called " depletion " of the mast cells of the liver ;
Thirdly , the method of stabilizing liver mast cells and compound 48 / 80 depleted liver mast cells by sodium cromoglycate can reduce the degree of liver ischemia reperfusion injury , which is reflected by the Suzuki score , the ALT and AST in the serum , the MDA of the liver tissue lysate , and the like .

Conclusion : Mast cell degranulation promotes ischemia - reperfusion injury in rat liver .

The effect of the second part on the survival of the receptor after orthotopic liver transplantation in rats and its mechanism

Background : Immunosuppressive therapy after transplantation can lead to the infection of opportunistic bacteria and the formation of tumor . Our previous studies have shown that by stabilizing mast cells and compound 48 / 80 " depleted " mast cells by sodium crotalate , the ischemia - reperfusion injury of the liver can be reduced . This part is intended to investigate the effects of these two methods of regulating mast cells on the survival of the receptor and its mechanism .

Materials and Methods : A rat orthotopic liver transplantation model was established by stabilizing liver mast cells ( CRM group ) and compound 48 / 80 ( CMP48 / 80 ) with sodium cromoglycate . The model of orthotopic liver transplantation was established by using male DA rats as donor and female Lewis rats as donor and female Lewis rats as recipients . Male Lewis was used as donor and female Lewis as donor and female Lewis as negative pair ( Syn group ) .

Results : Compared with PBS group , the survival time of CRM and CMP48 / 80 group was significantly prolonged compared with PBS group ( 17.40 鹵 1.67 and 14.20 鹵 2.28 days respectively ) , and the rejection activity index and liver function were significantly improved ( p 0.05 ) . The expression of IFN - 緯 in liver tissue and the level of protein in serum decreased significantly in Th1 .
and Th2 represents the expression of IL - 10 in liver tissue and the level of protein in serum .

Conclusion : It is possible to stabilize the Th1 / Th2 immune balance in the Th2 direction by stabilizing the leading status of liver mast cells and compound 48 / 80 depleted donor liver mast cells by sodium crotalate . This results in a possible reduction in the Th1 / Th2 immune balance in the Th2 direction .

Study on the effect and mechanism of the third partial transplantation of the mast cells regulating the receptor on acute rejection

Background : The role of mast cells in acute rejection is not very clear in the study . The aim of this study is to investigate the effect of post - transplantation receptor mast cell degranulation and its effect on acute rejection .

Materials and Methods : In the in vitro experimental part , we isolated rat primary skin mast cells ( RSMC ) and peritoneal mast cells ( RPMC ) , then respectively detected the expression of the surface receptor C5a of mast cells and their ability to degranulation under the action of C5a .
In vivo experiments , we established a model of ectopic heart transplantation from DA rats to Lewis rats . The groups were designed according to the receptor treatment methods : the PBS group injected with PBS , the CRM group injected with sodium glycate , the CsA3 + CRM group injected with 3 mg / kg body weight CsA and the CsA3 + CRM group injected with 5 mg / kg body weight , and the same gene transplantation group . The survival time of each group was observed .
the mast cell degranulation kit is used for detecting the degranulation level of the mast cell ;
The transplanted heart was stained with HE and acute rejection was observed .
The mRNA was extracted from the transplanted heart tissue , and the transcription level of IL - 10 and IFN - 緯 was detected .

Results : In vitro study , the expression of C5a receptor was 58.69 % ~ 67.64 % . The C5a receptor was only 1.23 % ~ 1.89 % of RPMC . C5a could induce RSMC and LPS - stimulated release of RPMC . C5a could reduce the level of degranulation of receptor mast cells . However , there was no significant difference in the levels of IL - 10 , IL - 10 , IFN - 緯 mRNA and IL - 10 and IFN - 緯 in the heart rejection score , cardiac tissue IL - 10 , IFN - 緯 mRNA and serum .

Conclusion : C5a and mast cell degranulation levels increased in rats with ectopic heart transplantation .
C5a can also promote the degranulation of mast cells .
Sodium glycate can reduce the degree of degranulation of mast cells , but it is not possible to reduce acute rejection and prolong graft survival .

【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R657.3

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相關(guān)期刊論文 前6條

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本文編號(hào)：1853349