【摘要】：[研究背景] 器官移植后必需應(yīng)用免疫抑制劑防止急性排斥反應(yīng)的發(fā)生,免疫抑制劑通過抑制T細(xì)胞和B細(xì)胞等靶細(xì)胞的細(xì)胞增殖、細(xì)胞因子合成而起作用。但是目前仍然無法區(qū)分移植抗原引起的免疫排斥反應(yīng)和病毒細(xì)菌感染引起的免疫反應(yīng)。理想的免疫抑制治療應(yīng)該在免疫抑制和保持免疫力間取得平衡,因此對(duì)于臨床醫(yī)生來說移植患者管理是一件很困難的事。保存受體免疫力,盡可能減小免疫抑制劑用量或誘導(dǎo)免疫耐受一直是移植免疫學(xué)家的主要目標(biāo),這方面雖然也取得了一些令人興奮的成果,但是誘導(dǎo)耐受仍然有大量的工作要做,研究誘導(dǎo)免疫耐受方法,盡可能減小免疫抑制的用量,研究檢測免疫耐受及免疫力的免疫學(xué)方法將是今后幾年器官移植所面臨的最重要的挑戰(zhàn)。 可靠的免疫狀態(tài)指標(biāo)可以為免疫抑制劑的使用提供依據(jù),使抑制劑不僅可以根據(jù)排斥反應(yīng)的程度或耐受情況使用,還可以根據(jù)患者的易感性規(guī)范化用藥,由于長期免疫抑制劑治療所引起的的高發(fā)病率和高死亡率,高度期望規(guī)范化用藥能夠?qū)崿F(xiàn)。但是,盡管通過血液標(biāo)本或移植物病理分析檢測免疫功能已經(jīng)可以部分了解宿主抗移植物排斥反應(yīng),包括了解浸潤細(xì)胞的表型及功能,T細(xì)胞性質(zhì)的改變、細(xì)胞因子及抗體的合成情況等,但是仍然沒有一種可靠的特異的排斥反應(yīng)檢測方法。目前臨床常用受體免疫狀態(tài)的監(jiān)測的方式仍然是臨床癥狀評(píng)估結(jié)合移植物活檢。但是,活組織病理檢查對(duì)移植物和宿主均是一種損傷性檢查,有其固有的局限性。 現(xiàn)在有學(xué)者開始供體特異性體內(nèi)細(xì)胞毒性試驗(yàn)的研究,用帶有移植物復(fù)合抗原的供體細(xì)胞檢測受體的免疫殺傷強(qiáng)度,供體細(xì)胞在受體內(nèi)受到的不僅是毒性淋巴細(xì)胞的攻擊,而且同時(shí)要受到抗體、補(bǔ)體、毒性淋巴細(xì)胞的多重攻擊,因此對(duì)移植復(fù)合抗原的定量測試不但能反映特異性CTL的活性,而且能全面的反映受體對(duì)供體的特異性免疫狀態(tài)。 本課題設(shè)計(jì)了一種簡單的方法,在新西蘭白兔上開展供體特異性體內(nèi)細(xì)胞毒性試驗(yàn),探索兔體內(nèi)細(xì)胞毒性試驗(yàn)方法建立的技術(shù)參數(shù),研究體內(nèi)細(xì)胞毒性試驗(yàn)?zāi)芊駲z測出受體的免疫排斥狀況。用CFSE標(biāo)記供體脾細(xì)胞,流式細(xì)胞儀檢測供體細(xì)胞在受體內(nèi)遭受排斥反應(yīng)過程,同時(shí)設(shè)置受體脾細(xì)胞作為內(nèi)參照。標(biāo)記的脾細(xì)胞懸液輸注給不同的受體,輸注后不同時(shí)間點(diǎn)采血進(jìn)行流式細(xì)胞分析。我們發(fā)現(xiàn)外周血中標(biāo)記的受體細(xì)胞能在8小時(shí)內(nèi)被流式細(xì)胞儀檢測得到,而不同的主要和次要組織相容性抗原的供體細(xì)胞在試驗(yàn)中消失與皮膚移植后排斥動(dòng)力學(xué)相一致。 [目的] 建立新西蘭白兔供體特異性體內(nèi)細(xì)胞毒性試驗(yàn)方法,探索細(xì)胞毒性試驗(yàn)檢測的技術(shù)參數(shù)、羥基熒光素乙酰乙酸琥珀酰亞胺酯(CFSE)熒光染色條件、輸注細(xì)胞數(shù)及確定最佳測試時(shí)間點(diǎn),驗(yàn)證體內(nèi)細(xì)胞毒性實(shí)驗(yàn)?zāi)芊駲z測出受體的免疫排斥狀況,為供體特異性體內(nèi)細(xì)胞毒性試驗(yàn)的臨床應(yīng)用提供新的依據(jù)。 [方法] 取4-6月齡清潔級(jí)雌、雄新西蘭白兔各5只,雌、雄兔各根據(jù)體重排序,根據(jù)序號(hào)雌雄兔配對(duì),雌兔為供體,雄兔為受體,于每只雌兔背部剪下約4cm×2cm的皮膚一塊,于雄兔背部移植雌兔皮膚,并用3/0無創(chuàng)傷縫線固定皮緣,移植后2周,移植皮膚完全脫落,建立新西蘭白兔雌-雄兔皮膚移植模型,為模型組。另取未作移植的雌、雄新西蘭白兔各5只,雌、雄兔各根據(jù)體重排序,根據(jù)序號(hào)雌雄兔配對(duì),為對(duì)照組。分別于模型組及對(duì)照組配對(duì)取雌兔及雄兔脾臟,放入Hank's液中,200目不銹鋼網(wǎng)研磨法制備脾細(xì)胞懸液,7.5g/L氯化銨裂解紅細(xì)胞,0.01mol/L PBS稀釋制備脾細(xì)胞單細(xì)胞懸液。雄性脾細(xì)胞用0.24μm CFSE染色,雌性脾細(xì)胞用高濃度6μm CFSE染色。每對(duì)雌、雄兔脾細(xì)胞懸液混合,制備1：1混合細(xì)胞懸液。細(xì)胞混合懸液稀釋后,取1滴稀釋液和1滴0.5%錐蟲藍(lán)溶液混合,靜置3min,加至細(xì)胞計(jì)數(shù)板,倒置顯微鏡下觀察,活細(xì)胞不著色,死細(xì)胞核成藍(lán)色,計(jì)數(shù)活細(xì)胞和死細(xì)胞數(shù)量,活細(xì)胞比例達(dá)95%以上(細(xì)胞存活率=0.5%錐蟲藍(lán)染色陰性細(xì)胞/100個(gè)細(xì)胞/HP×100)。于模型組、對(duì)照組雄免輸注細(xì)胞懸液,注射混合細(xì)胞總數(shù)為1×109個(gè)左右。雄兔輸注混合細(xì)胞后分別于1,2,4,8h抽取外周血,流式細(xì)胞儀檢測外周血兩種熒光細(xì)胞的比例變化,計(jì)算供體特異性細(xì)胞殺傷率(R)=1-donor cells/recipient cells×100%,比較模型組與對(duì)照組的供體特異性細(xì)胞殺傷率差異。實(shí)驗(yàn)數(shù)據(jù)應(yīng)用SPSS13.0軟件進(jìn)行統(tǒng)計(jì)學(xué)分析,所有數(shù)據(jù)示為x±s,組間殺傷率比較用t檢驗(yàn),P0.05認(rèn)為差異有顯著性意義。 [結(jié)果] 1.實(shí)驗(yàn)動(dòng)物數(shù)量分析模型組及對(duì)照組新西蘭白兔各10只,每組分別5對(duì)雌、雄兔。全部進(jìn)入結(jié)果分析,無脫失。 2.皮膚移植模型觀察結(jié)果皮膚移植后15d,移植皮膚完全脫落,雌-雄兔皮膚移植排斥模型建立,皮膚病理證實(shí)。 3. CFSE染色混合脾細(xì)胞懸液集中在淋巴細(xì)胞所在的小細(xì)胞區(qū),單核細(xì)胞區(qū)也有一部分陽性細(xì)胞,但是染色不均一,選用小淋巴細(xì)胞區(qū)作為檢測門。CFSE染色時(shí)需要較大濃度差才能完全把兩群脾細(xì)胞完全分離開,在濃度相差25倍時(shí)流式圖才顯示為兩組獨(dú)立的點(diǎn)圖,即CFSE低濃度用0.24μM,高濃度用6μM。 4.流式細(xì)胞學(xué)分析結(jié)果CFSE染色后的混合細(xì)胞在輸注前供體、受體細(xì)胞各為49.79%及49.85%。移植模型組供體雌兔脾細(xì)胞在輸注后1h剩4.73%,在2-4h后剩0.30%,8h后基本清除,而同基因的雄兔脾細(xì)胞同對(duì)照組一樣緩慢下降。模型組供體特異性細(xì)胞殺傷率(R)在混合細(xì)胞輸注后1h為48.26%+2.31%,2h為64.18%±6.23%,4h為73.41%±3.87%,8h為89.66%±6.96%。而對(duì)照組殺傷率在混合細(xì)胞輸注后1h為5.49%±2.51%,2h為13.23%±2.23%,4h為21.53%±2.23%,8h為31.51%±3.21%。在混合細(xì)胞輸注后的1h、2h、4h、8h,各時(shí)間點(diǎn)模型組供體特異性細(xì)胞殺傷率(R)明顯高于對(duì)照組,差異有統(tǒng)計(jì)學(xué)意義(t=25.284,13.624,24.215,15.146,所有P0.01)。 [結(jié)論] 新西蘭雌-雄兔皮膚移植模型體內(nèi)抗原特異性細(xì)胞毒性試驗(yàn)流式檢測設(shè)定在淋巴細(xì)胞門,兩種細(xì)胞CFSE的染色濃度差控制在25倍左右,輸注細(xì)胞1,2h模型組殺傷率高達(dá)48.26%±2.31%,64.18%±6.23%,選擇這一時(shí)間點(diǎn)可以準(zhǔn)確判定兩組白兔所處的免疫狀態(tài),且能滿足臨床要求的快速檢測的目的。供體特異性體內(nèi)細(xì)胞毒性試驗(yàn)的特點(diǎn)有： 1、供體特異性體內(nèi)細(xì)胞毒性試驗(yàn)是體內(nèi)試驗(yàn),不用模擬體內(nèi)復(fù)雜的免疫環(huán)境,本身就是移植物所處的免疫環(huán)境。 2、檢測方法準(zhǔn)確簡單,應(yīng)用熒光標(biāo)記流式細(xì)胞技術(shù)檢測,可準(zhǔn)確到單個(gè)細(xì)胞水平。 3、檢測靶細(xì)胞為供體來源細(xì)胞(脾細(xì)胞),抗原性與移植物相同,為復(fù)合抗原,容易獲得,能準(zhǔn)確反映移植物所受免疫攻擊情況。 4、內(nèi)參照細(xì)胞為受體來源同基因細(xì)胞(脾細(xì)胞),控制了試驗(yàn)偶然誤差和系統(tǒng)誤差；內(nèi)參照細(xì)胞在體外處理及體內(nèi)免疫反應(yīng)的全過程始終與試驗(yàn)靶細(xì)胞處于同樣的反應(yīng)體系中,消除了由于操作及體內(nèi)非特異性反應(yīng)所致的誤差。
[Abstract]:BACKGROUND OF THE STUDY

Immunosuppressive therapy should be used to prevent acute rejection after organ transplantation , and the immune inhibitor can play a role in inhibiting cell proliferation and cytokine synthesis of target cells such as T cells and B cells . However , it is difficult to differentiate between immune rejection caused by transplantation antigen and immune response caused by virus bacterial infection .

The reliable immune status indicator can provide a basis for the use of an immunosuppressive agent , so that the inhibitor can be used not only according to the degree or tolerance of the rejection reaction , but also can be realized according to the susceptibility of the patient .

At present , some scholars begin to study the cytotoxicity test of donor - specific in vivo , and use donor cells with graft composite antigen to detect the immune killing intensity of the receptor . The donor cells are not only attacked by toxic lymphocytes in the body , but also can be subjected to multiple attacks of antibodies , complement and toxic lymphocytes , so that the quantitative test of the grafted composite antigen can not only reflect the activity of the specific CTL , but also can comprehensively reflect the specific immune status of the receptor to the donor .

In this study , a simple method was designed to study the cytotoxicity of donor - specific in vivo in New Zealand white rabbits . The technique parameters established in vivo cytotoxicity test were explored . The donor spleen cells were labeled with CFSE . The donor spleen cells were detected by flow cytometry .

Purpose of the project

New Zealand white rabbit donor specific in vivo cytotoxicity test method was established to explore the technical parameters of cytotoxicity test , the fluorescent staining conditions of hydroxyfluorescein acetoacetic acid succinimide ester ( CFSE ) , the number of infusion cells and the determination of the optimal test time point , to verify whether the in vivo cytotoxicity test can detect the immune rejection condition of the receptor , and provide a new basis for clinical application of the donor specific in vivo cytotoxicity test .

Methodology

Male and female rabbits were divided into 4 - 6 months old female and male New Zealand white rabbits according to their weight . The male and female rabbits were divided into two groups according to the weight of the male and female rabbits .

The result is not valid .

1 . There were 10 rabbits in the model group and control group , 5 pairs of female and male rabbits in each group . All the results were analyzed without loss of loss .

2 . After skin transplantation , 15 days after skin transplantation , the skin was completely peeled off , and the female - male rabbit skin graft rejection model was established , and the skin pathology was confirmed .

3 . CFSE staining mixed spleen cell suspension concentrated in the small cell area in which the lymphocytes were located , but the mononuclear cell area also had some positive cells , but the staining was not uniform , and the small lymphocyte region was used as the detection gate . When CFSE staining , the two groups of spleen cells were completely separated , and the flow graph was shown as two groups of independent point graphs at 25 times the concentration of CFSE , namely , the concentration of CFSE was 0.24 . m u.M and the high concentration was 6 . m

4 . The results of flow cytometry analysis showed that the donor and recipient cells were 49.79 % and 49.85 % after the infusion of the mixed cells . The total cell killing rate ( R ) of the donor - specific cells in the model group was 48.26 % + 2.31 % , 2h was 64.18 % 鹵 6.23 % , 2h was 13.23 % 鹵 3.87 % , 8h was 89.66 % 鹵 6.96 % . The difference was statistically significant ( t = 25.284 , 13.624 , 24.215 , 15.146 , all P0.01 ) .

Conclusion

in New Zealand female - male rabbit skin transplantation model in vivo antigen - specific cytotoxicity assay was set up in lymphocyte door , and that difference of staining concentration of two cell CFSE was controlled at about 25 times , and the killing rate of the two groups of cells was up to 48.26 % 鹵 2.31 % , 64.18 % 鹵 6.23 % .

1 . The cytotoxicity test of donor - specific in - vivo cytotoxicity test is in vivo test without simulating the complex immune environment in vivo , which is the immune environment at which the graft is located .

and 2 , the detection method is accurate and simple , and the detection of the fluorescence labeled flow cytometry can be applied to the single cell level accurately .

3 . The detection target cells are donor - derived cells ( spleen cells ) , the antigenicity of which is the same as that of the graft , is a composite antigen , is easy to obtain , and can accurately reflect the immune attack condition of the graft .

4 . The internal reference cell is the receptor - derived co - gene cell ( spleen cell ) , which controls the experimental error and systematic error .
The internal reference cell is always in the same reaction system as the test target cell in the whole process of in vitro treatment and in vivo immune response , and the error caused by the operation and the nonspecific reaction in vivo is eliminated .
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392

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