中华器官移植杂志
中華器官移植雜誌
중화기관이식잡지
CHINESE JOURNAL OF ORGAN TRANSPLANTATION
2014年
5期
300-305
,共6页
王璐%王军祥%王筱啸%陈松%陈刚
王璐%王軍祥%王篠嘯%陳鬆%陳剛
왕로%왕군상%왕소소%진송%진강
肾皮质%上皮细胞%ABO血型系统%肾移植%流式细胞术
腎皮質%上皮細胞%ABO血型繫統%腎移植%流式細胞術
신피질%상피세포%ABO혈형계통%신이식%류식세포술
Kidney cortex%Epithelial cells%ABO Blood-group system%Kidney transplantation%Flow cytometry
目的 探讨以食蟹猴皮质区肾小管上皮细胞(RCTEC)为靶细胞检测ABO血型抗体滴度的可行性.方法 分离培养食蟹猴原代RCTEC,蛋白质印迹法和流式细胞术鉴定细胞的表型与纯度.利用流式细胞术分别检测ABO组织-血型抗原在RCTEC上的表达、RCTEC与人血清血型抗体的结合能力以及比较人红细胞与RCTEC测定血型抗体滴度的差异.结果 分离得到的肾皮质细胞绝大部分是RCTEC.39.90％～73.80％的RCTEC表达ABO组织-血型抗原,且能与人血清中的血型抗体特异性结合.较长时间冻存对RCTEC的ABO组织-血型抗原表达水平无明显影响,冻存前表达B型抗原的阳性细胞比例为39.90％,冻存10个月后复苏再培养9d,阳性细胞的比例为39..65％.利用钥孔戚血蓝蛋白偶联的血型抗原A(KLH-A)致敏前后的食蟹猴血清对比原代RCTEC与人红细胞检测血型抗体滴度的效果,结果显示,在RCTEC检测组,未出现凝集现象,与两种血清中抗体结合的几何平均荧光强度(MFI)均在最低血清稀释度(1∶16)时最高,其后均随血清稀释度的增加而逐渐降低,未致敏血清的MFI值在稀释度为1∶128时降至基线水平,致敏血清的MFI值在1∶8 192稀释度时降至基线水平,在1∶16～1∶8 192稀释度之间,未致敏血清的MFI值均低于致敏血清;在人红细胞检测组,当抗体滴度高时凝集现象明显,未致敏血清MFI值的最高峰出现在1∶64稀释度时,致敏血清MFI值的最高峰出现在1∶32稀释度时,且随血清稀释度的增加呈现无明显规律的波动,在1∶64～1∶512稀释度之间,未致敏血清MFI值高于致敏血清.利用RCTEC检测血型抗体滴度的MFI值能较准确地反映抗体滴度的变化.结论 食蟹猴RCTEC高表达ABO组织-血型抗原,比人红细胞更适用于血清中血型抗体滴度的流式细胞术检测,具有良好的临床与科研应用潜力.
目的 探討以食蟹猴皮質區腎小管上皮細胞(RCTEC)為靶細胞檢測ABO血型抗體滴度的可行性.方法 分離培養食蟹猴原代RCTEC,蛋白質印跡法和流式細胞術鑒定細胞的錶型與純度.利用流式細胞術分彆檢測ABO組織-血型抗原在RCTEC上的錶達、RCTEC與人血清血型抗體的結閤能力以及比較人紅細胞與RCTEC測定血型抗體滴度的差異.結果 分離得到的腎皮質細胞絕大部分是RCTEC.39.90％～73.80％的RCTEC錶達ABO組織-血型抗原,且能與人血清中的血型抗體特異性結閤.較長時間凍存對RCTEC的ABO組織-血型抗原錶達水平無明顯影響,凍存前錶達B型抗原的暘性細胞比例為39.90％,凍存10箇月後複囌再培養9d,暘性細胞的比例為39..65％.利用鑰孔慼血藍蛋白偶聯的血型抗原A(KLH-A)緻敏前後的食蟹猴血清對比原代RCTEC與人紅細胞檢測血型抗體滴度的效果,結果顯示,在RCTEC檢測組,未齣現凝集現象,與兩種血清中抗體結閤的幾何平均熒光彊度(MFI)均在最低血清稀釋度(1∶16)時最高,其後均隨血清稀釋度的增加而逐漸降低,未緻敏血清的MFI值在稀釋度為1∶128時降至基線水平,緻敏血清的MFI值在1∶8 192稀釋度時降至基線水平,在1∶16～1∶8 192稀釋度之間,未緻敏血清的MFI值均低于緻敏血清;在人紅細胞檢測組,噹抗體滴度高時凝集現象明顯,未緻敏血清MFI值的最高峰齣現在1∶64稀釋度時,緻敏血清MFI值的最高峰齣現在1∶32稀釋度時,且隨血清稀釋度的增加呈現無明顯規律的波動,在1∶64～1∶512稀釋度之間,未緻敏血清MFI值高于緻敏血清.利用RCTEC檢測血型抗體滴度的MFI值能較準確地反映抗體滴度的變化.結論 食蟹猴RCTEC高錶達ABO組織-血型抗原,比人紅細胞更適用于血清中血型抗體滴度的流式細胞術檢測,具有良好的臨床與科研應用潛力.
목적 탐토이식해후피질구신소관상피세포(RCTEC)위파세포검측ABO혈형항체적도적가행성.방법 분리배양식해후원대RCTEC,단백질인적법화류식세포술감정세포적표형여순도.이용류식세포술분별검측ABO조직-혈형항원재RCTEC상적표체、RCTEC여인혈청혈형항체적결합능력이급비교인홍세포여RCTEC측정혈형항체적도적차이.결과 분리득도적신피질세포절대부분시RCTEC.39.90％～73.80％적RCTEC표체ABO조직-혈형항원,차능여인혈청중적혈형항체특이성결합.교장시간동존대RCTEC적ABO조직-혈형항원표체수평무명현영향,동존전표체B형항원적양성세포비례위39.90％,동존10개월후복소재배양9d,양성세포적비례위39..65％.이용약공척혈람단백우련적혈형항원A(KLH-A)치민전후적식해후혈청대비원대RCTEC여인홍세포검측혈형항체적도적효과,결과현시,재RCTEC검측조,미출현응집현상,여량충혈청중항체결합적궤하평균형광강도(MFI)균재최저혈청희석도(1∶16)시최고,기후균수혈청희석도적증가이축점강저,미치민혈청적MFI치재희석도위1∶128시강지기선수평,치민혈청적MFI치재1∶8 192희석도시강지기선수평,재1∶16～1∶8 192희석도지간,미치민혈청적MFI치균저우치민혈청;재인홍세포검측조,당항체적도고시응집현상명현,미치민혈청MFI치적최고봉출현재1∶64희석도시,치민혈청MFI치적최고봉출현재1∶32희석도시,차수혈청희석도적증가정현무명현규률적파동,재1∶64～1∶512희석도지간,미치민혈청MFI치고우치민혈청.이용RCTEC검측혈형항체적도적MFI치능교준학지반영항체적도적변화.결론 식해후RCTEC고표체ABO조직-혈형항원,비인홍세포경괄용우혈청중혈형항체적도적류식세포술검측,구유량호적림상여과연응용잠력.
Objective To investigate the possibility of measuring ABO blood group antibody levels using renal cortical tubular epithelial cells (RCTECs) of cynomolgus monkey.Method The primary RCTECs were isolated from cynomolgus monkey kidneys and identified by Western blotting and flow cytometry (FACS).FACS was applied to detect the expression of ABO histo-blood group antigens on RCTECs,the binding of blood group antibodies in human serum to RCTECs,and to compare the difference of measuring ABO blood group antibody levels between using human erythrocytes and RCTECs as target cells.Result The majority of cells derived from the kidney cortex were renal tubular epithelial cells.39.90％-73.80％ of RCTECs were found to express the ABO histoblood group antigens with the capability to bind the human blood group antibodies specifically.The expression level of ABO histo-blood group antigen on RCTECs was not influenced by long-term cryopreservation,and the percentage of B antigen positive cells was 39.90％ before frozen storage and 39.65％ after a 10-month cryopreservation respectively.The normal serum and allergic serum presensitized to synthetic A antigen-keyhole limpet hemocyanin (KLH-A) of cynomolgus monkey were used to compare the abilities of RCTECs and human erythrocytes to detect antibody titers.No agglutination was observed in RCTECs test group.The mean fluorescence intensity (MFI) values of antibody were highest when the dilution fold was lowest (1 ∶ 16) and gradually decreased with increased serum dilution in both serum groups.MFI fell towards baseline value at 1 ∶ 128 dilution in normal serum group while at 1∶8 192 dilution in allergic serum group.Between 1 ∶ 16 and 1∶8 192 dilutions,MFIs of normal serum group were all lower than those of allergic serum.In human red cells test group,obvious agglutination appeared at high concentrations of antibodies,and MFIs reached the peak at 1∶64 dilution in normal serum group and at 1∶32 dilution in allergic serum group,but fluctuated irregularly thereafter.Between 1∶64 and 1∶512 dilutions,MFIs of normal serum group were all higher than those of allergic serum.Data above showed that MFI values in RCTECs group could reflect the levels of blood group antibodies more exactly.Conclusion The RCTECs of cynomolgus monkey express ABO tissue-blood group antigens and would be more suitable to be used to measure ABO blood group antibody levels by FACS.
