新西蘭雌—雄兔皮膚移植模型供體特異性體內(nèi)細胞毒性試驗方法的建立
發(fā)布時間：2018-07-16 16:02
【摘要】：[研究背景] 器官移植后必需應用免疫抑制劑防止急性排斥反應的發(fā)生,免疫抑制劑通過抑制T細胞和B細胞等靶細胞的細胞增殖、細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和病毒細菌感染引起的免疫反應。理想的免疫抑制治療應該在免疫抑制和保持免疫力間取得平衡,因此對于臨床醫(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免疫耐受一直是移植免疫學家的主要目標,這方面雖然也取得了一些令人興奮的成果,但是誘導耐受仍然有大量的工作要做,研究誘導免疫耐受方法,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,包括了解浸潤細胞的表型及功能,T細胞性質(zhì)的改變、細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估結合移植物活檢。但是,活組織病理檢查對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)在有學者開始供體特異性體內(nèi)細胞毒性試驗的研究,用帶有移植物復合抗原的供體細胞檢測受體的免疫殺傷強度,供體細胞在受體內(nèi)受到的不僅是毒性淋巴細胞的攻擊,而且同時要受到抗體、補體、毒性淋巴細胞的多重攻擊,因此對移植復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供體的特異性免疫狀態(tài)。 本課題設計了一種簡單的方法,在新西蘭白兔上開展供體特異性體內(nèi)細胞毒性試驗,探索兔體內(nèi)細胞毒性試驗方法建立的技術參數(shù),研究體內(nèi)細胞毒性試驗能否檢測出受體的免疫排斥狀況。用CFSE標記供體脾細胞,流式細胞儀檢測供體細胞在受體內(nèi)遭受排斥反應過程,同時設置受體脾細胞作為內(nèi)參照。標記的脾細胞懸液輸注給不同的受體,輸注后不同時間點采血進行流式細胞分析。我們發(fā)現(xiàn)外周血中標記的受體細胞能在8小時內(nèi)被流式細胞儀檢測得到,而不同的主要和次要組織相容性抗原的供體細胞在試驗中消失與皮膚移植后排斥動力學相一致。 [目的] 建立新西蘭白兔供體特異性體內(nèi)細胞毒性試驗方法,探索細胞毒性試驗檢測的技術參數(shù)、羥基熒光素乙酰乙酸琥珀酰亞胺酯(CFSE)熒光染色條件、輸注細胞數(shù)及確定最佳測試時間點,驗證體內(nèi)細胞毒性實驗能否檢測出受體的免疫排斥狀況,為供體特異性體內(nèi)細胞毒性試驗的臨床應用提供新的依據(jù)。 [方法] 取4-6月齡清潔級雌、雄新西蘭白兔各5只,雌、雄兔各根據(jù)體重排序,根據(jù)序號雌雄兔配對,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)序號雌雄兔配對,為對照組。分別于模型組及對照組配對取雌兔及雄兔脾臟,放入Hank's液中,200目不銹鋼網(wǎng)研磨法制備脾細胞懸液,7.5g/L氯化銨裂解紅細胞,0.01mol/L PBS稀釋制備脾細胞單細胞懸液。雄性脾細胞用0.24μm CFSE染色,雌性脾細胞用高濃度6μm CFSE染色。每對雌、雄兔脾細胞懸液混合,制備1：1混合細胞懸液。細胞混合懸液稀釋后,取1滴稀釋液和1滴0.5%錐蟲藍溶液混合,靜置3min,加至細胞計數(shù)板,倒置顯微鏡下觀察,活細胞不著色,死細胞核成藍色,計數(shù)活細胞和死細胞數(shù)量,活細胞比例達95%以上(細胞存活率=0.5%錐蟲藍染色陰性細胞/100個細胞/HP×100)。于模型組、對照組雄免輸注細胞懸液,注射混合細胞總數(shù)為1×109個左右。雄兔輸注混合細胞后分別于1,2,4,8h抽取外周血,流式細胞儀檢測外周血兩種熒光細胞的比例變化,計算供體特異性細胞殺傷率(R)=1-donor cells/recipient cells×100%,比較模型組與對照組的供體特異性細胞殺傷率差異。實驗數(shù)據(jù)應用SPSS13.0軟件進行統(tǒng)計學分析,所有數(shù)據(jù)示為x±s,組間殺傷率比較用t檢驗,P0.05認為差異有顯著性意義。 [結果] 1.實驗動物數(shù)量分析模型組及對照組新西蘭白兔各10只,每組分別5對雌、雄兔。全部進入結果分析,無脫失。 2.皮膚移植模型觀察結果皮膚移植后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。 3. CFSE染色混合脾細胞懸液集中在淋巴細胞所在的小細胞區(qū),單核細胞區(qū)也有一部分陽性細胞,但是染色不均一,選用小淋巴細胞區(qū)作為檢測門。CFSE染色時需要較大濃度差才能完全把兩群脾細胞完全分離開,在濃度相差25倍時流式圖才顯示為兩組獨立的點圖,即CFSE低濃度用0.24μM,高濃度用6μM。 4.流式細胞學分析結果CFSE染色后的混合細胞在輸注前供體、受體細胞各為49.79%及49.85%。移植模型組供體雌兔脾細胞在輸注后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胞同對照組一樣緩慢下降。模型組供體特異性細胞殺傷率(R)在混合細胞輸注后1h為48.26%+2.31%,2h為64.18%±6.23%,4h為73.41%±3.87%,8h為89.66%±6.96%。而對照組殺傷率在混合細胞輸注后1h為5.49%±2.51%,2h為13.23%±2.23%,4h為21.53%±2.23%,8h為31.51%±3.21%。在混合細胞輸注后的1h、2h、4h、8h,各時間點模型組供體特異性細胞殺傷率(R)明顯高于對照組,差異有統(tǒng)計學意義(t=25.284,13.624,24.215,15.146,所有P0.01)。 [結論] 新西蘭雌-雄兔皮膚移植模型體內(nèi)抗原特異性細胞毒性試驗流式檢測設定在淋巴細胞門,兩種細胞CFSE的染色濃度差控制在25倍左右,輸注細胞1,2h模型組殺傷率高達48.26%±2.31%,64.18%±6.23%,選擇這一時間點可以準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)細胞毒性試驗的特點有： 1、供體特異性體內(nèi)細胞毒性試驗是體內(nèi)試驗,不用模擬體內(nèi)復雜的免疫環(huán)境,本身就是移植物所處的免疫環(huán)境。 2、檢測方法準確簡單,應用熒光標記流式細胞技術檢測,可準確到單個細胞水平。 3、檢測靶細胞為供體來源細胞(脾細胞),抗原性與移植物相同,為復合抗原,容易獲得,能準確反映移植物所受免疫攻擊情況。 4、內(nèi)參照細胞為受體來源同基因細胞(脾細胞),控制了試驗偶然誤差和系統(tǒng)誤差；內(nèi)參照細胞在體外處理及體內(nèi)免疫反應的全過程始終與試驗靶細胞處于同樣的反應體系中,消除了由于操作及體內(nèi)非特異性反應所致的誤差。
[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 .
【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R392
本文編號：2126898
[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 .
【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R392
【參考文獻】
相關期刊論文 前3條
1 張明明;周佩軍;邵琨;王祥慧;徐達;;轉移性體內(nèi)遲發(fā)型超敏反應監(jiān)測法的建立及臨床應用[J];上海交通大學學報(醫(yī)學版);2010年09期
2 蔣澤生;高毅;;熒光染料CFSE差異濃度標記活細胞體內(nèi)示蹤方法[J];中國組織工程研究與臨床康復;2010年15期
3 蔣澤生;高毅;;供體特異性體內(nèi)免疫狀態(tài)的定量檢測[J];中國組織工程研究與臨床康復;2010年31期
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