【作者】 李志赏；

【导师】 邵宗鸿；

【作者基本信息】 天津医科大学 ， 内科学， 2011， 硕士

【摘要】 目的分析重型再生障碍性贫血(SAA)患者免疫抑制治疗(IST)前、后外周血自然杀伤(NK)细胞及其亚群占外周血淋巴细胞百分比、功能变化及其与造血功能相关性,探讨NK细胞在SAA发病机制中的作用。方法用单克隆抗体四标法和流式细胞术(FCM)检测42例SAA患者[初治12例(初治组)、IST后恢复30例(恢复组)]外周血NK细胞(CD3CD56+/CD16+)及其亚群[CD56bright (CD3"CD56brightCD16neg/dim)、CD56dim (CD3-CD56dimCD16bnght)、CD3-CD56negCD16bright]占淋巴细胞的百分比、NK细胞胞膜抑制性受体(CD158a和CD158b)、NK细胞胞膜活化性受体(NKG2D和NKp46)及NK细胞胞浆穿孔素、颗粒酶B的表达,并与13名正常对照(对照组)做比较。分析上述变化与SAA患者外周血中性粒细胞百分比(ANC%)、淋巴细胞百分比(Lym%)、网织红细胞计数(Ret)及骨髓造血功能(增生程度、粒系百分比、红系百分比、巨核细胞数量、淋系百分比)的相关性。结果1.SAA患者外周血NK细胞及其亚群占淋巴细胞百分比的变化①SAA患者NK细胞百分比的变化初治组、恢复组、对照组NK细胞百分比分别为(10.30±6.08)%、(16.47±8.29)%、(19.45±6.88)%,初治组明显低于恢复组和对照组(均p<0.05),恢复组与对照组比较差异无统计学意义(p>0.05)②SAA患者CD56bnghtNK细胞亚群百分比的变化初治组、恢复组、对照组CD56bnght细胞亚群百分比的中位数分别为0.11%、0.68%、0.53%,初治组明显低于恢复组和对照组组(均p<0.05),恢复组与对照组比较差异无统计学意义(p>0.05)。③SAA患者CD56dim NK细胞亚群百分比的变化初治组、恢复组、对照组CD56dim NK细胞亚群百分比分别为(9.62±6.04)%、(13.81±7.52)%、(18.21±7.16)%,初治组明显低于对照组(p<0.05),初治组与恢复组比较差异无统计学意义(p>0.05),恢复组与对照组比较差异亦无统计学意义(p>0.05)。④SAA患者CD3-CD56negCD16bright NK细胞亚群百分比的变化初治组、恢复组、对照组CD3-CD56negCD16bright NK细胞亚群百分比的中位数分别为0.37%、0.79%、0.41%,恢复组明显高于初治组及对照组(均p<0.05),初治组与对照组比较差异无统计学意义(p>0.05)。2.SAA患者外周血NK细胞功能的变化①SAA患者NK细胞胞膜受体表达的变化SAA初治组NK细胞胞膜CD158a、NKG2D、NKp46中位表达阳性率分别为16.45%、95.68%、88.23%,SAA恢复组为19.34%、96.85%、82.97%,对照组为21.59%、96.13%、40.99%,初治组与恢复组NKp46表达阳性率明显高于对照组(均p<0.05)；初治组、恢复组、对照组NK细胞胞膜CD158b表达阳性率分别为(34.66±16.02)%、(39.13±17.75)%、(41.43±17.43)%,各组之间比较差异均无统计学意义(均p>0.05)。②SAA患者NK细胞胞浆穿孔素、颗粒酶B表达的变化初治组、恢复组、对照组NK细胞胞浆穿孔素表达阳性率分别为(64.97±21.61)%、(66.14±20.73)%、(42.11±27.25)%,初治组与恢复组均明显高于对照组(均p<0.05),初治组与恢复组比较差异无统计学意义(p>0.05)；初治组、恢复组、对照组NK细胞胞浆颗粒酶B表达的中位阳性率分别为96.43%、96.97%、92.88%,各组之间比较差异均无统计学意义(均p>0.05)。3.SAA患者外周血NK细胞及亚群占淋巴细胞百分比、功能与造血功能的相关性①SAA患者NK细胞及其亚群百分比与造血功能的相关性NK细胞百分比与SAA患者ANC%、骨髓增生程度、骨髓粒系及红系百分比呈正相关(r分别为0.423、0.455、0.357、0.340,均p<0.05),与Lymm%、骨髓淋系百分比呈负相关(r分别为-0.411、-0.522,均p<0.05)；CD56brightNK细胞亚群百分比与SAA患者ANC%、骨髓增生程度、骨髓的粒系百分比及巨核细胞数量呈关正相关(r分别为0.609、0.412、0.517、0.362,均p<0.05),与骨髓淋系百分比呈负相关(r=-0.435,p＜0.05); CD56dimNK细胞亚群百分比与SAA患者骨髓增生程度呈正相关(r=0.404,p<0.05),与骨髓淋系百分比呈负相关(r=-0.411,p<0.05)；CD3-CD56negCD16bright NK细胞亚群百分比与SAA患者ANC%、Ret、骨髓增生程度、骨髓粒系百分比呈正相关(r分别为0.468、0.328、0.451、0.434,均p<0.05),与Lymm%、骨髓淋系百分比呈负相关(r分别为-0.389、-0.547,均p<0.05)。②SAA患者NK细胞功能与造血功能的相关性NK细胞的CD158a、CD158b、NKG2D、NKp46、穿孔素、颗粒酶B表达与前述各造血功能指标均无相关性(均p>0.05)。结论SAA患者外周血NK细胞、CD56brigt、CD56dimNK细胞亚群占淋巴细胞百分比降低可能引起患者免疫耐受被破坏、T细胞功能亢进而导致造血功能衰竭；SAA患者外周血NK细胞NKp46、穿孔素表达增强可能是对NK细胞数量不足的一种“代偿”。更多还原

【Abstract】 ObjectiveTo investigate the quantitative and functional changes of natural killer (NK) cells and it’s subsets in peripheral blood of severe aplastic anemia (SAA) patients before and after immunosuppressive therapy (IST),evaluate the relationships between these changes and hematopoietic function,and explore the role of NK cells in the the pathogenisis of SAA.MethodsBy means of four-color monoclonal antibody labeling technology, the percentag-es of NK cells (CD3-CD56+/CD16+) and it’s subsets [CD56bright (CD3-CD56bright CD16neg/dim) CD56dim (CD3-CD56dimCD16bright),CD3-CD56negCD16bright] in peripheral blood were detected in 42 SAA patients,including 12 untreated patients and 30 recovered patients, and 13 normal controls respectively. NK cells inhibitory receptors (CD158a and CD158b), activating receptors (NKG2D and NKp46), perforrin and granzyme-βof patients and normal controls were also detected.The correlation between these changes and hematopoietic function, including percentage of neutron-phil granulocyte (ANC%), percentage of lymphocyte (Lym%) and reticulocyte absol-ute value (Ret) in peripheral blood,and hyperplasia degree,percentage of granulocytes, erythrocytes,lymphocytes and megakaryocytes absolute value in bone marrow were evaluated.Results1. The variations of NK cells and it’s subsets percentages in peripheral blood lymphocytes in SAA patients①The change in the proportion of NK cells in SAA patients The percentage of NK cells in peripheral blood lymphocytes was (10.30±6.08)% in untreated SAA patients, (16.47±8.29)% in recovered patients, and (19.45±6.88)% in normal controls. The percentage of NK cells in untreated SAA patients was lower than that of recovered patients and normal controls (p<0.05),but there was no statistical difference between recovered patients and normal controls (p>0.05).②The change in the proportion of CD56bright NK cells subset in SAA patients The median percentage of CD56bright NK cells subset was 0.11% in untreated SAA patients,0.68% in recovered patients and 0.53% in normal controls.The median percentage of CD56bnght cells in untreated patients was lower than that of the other two groups (p<0.05), but there was no statistical difference between recovered patients and normal controls (p>0.05).③The change in the proportion of CD56dim NK cells subset in SAA patients The percentage of CD56dim NK cells subset was (9.62±6.04)% in untreated patients, (13.81±7.52)% in recovered patients and (18.21±7.16)% in normal controls.The percentage of CD56dim cells in untreated SAA patients was lower than that of normal controls (p<0.05),but there was no statistical difference between the other groups (p>0.05).④The change in the proportion of CD3-CD56negCD16bright NK cells subset in SAA patients The median percentage of CD3-CD56negCD16bright NK cells subset was 0.37% in untreated SAA patients,0.79% in recovered patients and 0.41% in normal controls.The percentage of CD3-CD56negCD16bright NK cells subset in recovered SAA patients was higher than that of the other two groups (p<0.05), but there was no statistical difference between untreated patients and normal controls (p>0.05).2. The variation of NK cells function in SAA patients①The changes in the proportion of receptors on NK cells surface in SAA patients The median expressions of CD158a,NKG2D and NKp46 on NK cells surface were 16.45%,95.68%,88.23% in untreated SAA patients,19.34%,96.85%, 82.97% in recovered patients and 21.59%,96.13%,40.99% in normal controls.The expression of NKp46 on NK cells surface of untreated and recovered SAA patients were higher than that of healthy individuals (p<0.05).The expression of CD 158b on NK cells surface was (34.66±16.02)% in untreated patients,(39.13±17.75)% in recovered patients,(41.43±17.43)% in controls,but there was no statistical difference between each groups (p>0.05).②The changes in the proportion of perforin and granzyme-B in NK cells cytoplasm in SAA patients The expression of perforin in NK cells cytoplasm was (66.14±20.73)% in untreated SAA patients,(64.97±21.61)% in recovered patients and (42.11±27.25)% in controls.The expression of perforn in untreated and recovered SAA patients were higher than that of controls (p<0.05),but there was no statistical difference between that of the other groups (p>0.05).The median expression of granzyme-B in NK cells cytoplasm is 96.43% in untreated SAA patients,96.97% in recovered patients,92.88% in controls,there was no statistical difference between each groups (p>0.05).3. The correlations between the percentage of NK cells subsets and hematopoietic function in SAA patients①The correlations between the variations of NK cells and it’s subsets percenage and hematopoietic function in SAA patients The percentage NK cells was positively correlated with ANC%,hyperplasia degree of bone marrow,percentage of granuloc-ytes and erythrocytes in bone marrow (r was 0.423,0.455,0.357,0.340 respectively, p<0.05), but negatively correlated with Lym% in peripheral blood and percentage of lymphocytes in bone marrow(r was-0.411,-0.522 respectively, p<0.05).The percen-tage of CD56brightNK cells subset was positively correlated with ANC%, hyperplasia degree of bone marrow, percentage of granulocytes and megakaryocytes in bone marrow(r was 0.609,0.412,0.517,0.362 respectively,p＜0.05). Meanwhile, it was nega-tively correlated with the percentage of lymphocytes in bone marrow(r=-0.435, p<0.05).The percentage of CD56dim NK cells subset was positively correlated with hyperplasia degree of bone marrow (r=0.404, p<0.05), but negatively correlated with the percentage of lymphocytes in bone marrow (r=-0.411,p<0.05).The percentage of CD3-CD56negCD16bright NK cells subset was positively correlated with ANC%, Ret, hyperplasia degree of bone marrow, percentage of granulocytes in bone marrow (r was 0.468,0.328,0.451,0.434 respectively,p＜0.05).Yet, it was negatively correlated with Lym% in peripheral blood and the percentage of lymphocytes in bone marrow (r was-0.389,-0.547 respectively,p<0.05).②The correlation between the function of NK cells and hematopoietic function in SAA patients The expression of CD158a, CD158b, NKG2D, NKp46, perforrin and granzyme-βof NK cells has no correlation with hematopoiesis (p>0.05) in SAA patients.Conclusion The decrease in the percentage of NK cells, CD56bright, CD56dimNK cell subsets may have led to the over-function of T lymphocytes and the hematopoiesis failure in SAA. The higher expression of NKp46 and perforin of NK cells may be a compensation for the decrease of NK cells propotion.更多还原