单中心肾移植受者HLA抗原分布频率分析

目的分析肾移植受者HLA-A、B、DR和DQ抗原分布频率。 方法收集2017年1月至2018年6月在解放军总医院第八医学中心接受肾移植的791例受者HLA抗原检测数据,分析HLA-A、B、DR和DQ抗原种类和分布频率。 结果HLA-A抗原共出现18种,其中A2、A11和A24(9)抗原分布频率较高,分别为30.59%、17.83%和15.68%;A2403、A34(10)和A66(10)抗原分布频率最低,均为0.06%。HLA-B抗原共出现42种,其中分布频率最高的是B13(12.64%),分布频率最低的是B44、B41和40∶40(均为0.06%)。HLA-DR抗原共出现16种,其中DR15(2)、DR4和DR12(5)分布频率较高,分别为14.60%、13.65%和13.40%;DR6分布频率最低(0.19%);DR15(2)与DQ6(1)具有强连锁反应(90.48%)。HLA-DQ抗原共出现7种,DQ7(3)抗原分布频率最高(24.91%),DQ8(3)分布频率最低(5.50%)。 结论肾移植受者HLA-A、B、DR和DQ抗原中,A2、B13、DR15(2)和DQ7(3)分别为分布频率最高的抗原。分析HLA抗原分布频率有利于寻找HLA匹配的供者。     

肾移植供受者HLA—DR基因配型结果分析（附276例报告）

为了进一步提高肾移植水平，采用顺序特异引物聚合物反应（ＰＣＲ－ＳＳＰ）方法用于２７６例肾移植供受者ＨＬＡ－ＤＲ基因配型，以探讨其临床可行性和实用性，结果显示：２７６例供受者３０８份样本，ＰＣＲ－ＳＳＰ分型均获成功，检测时间５小时内，供受者ＤＲ等基在频分布分布大致相同，无显著性差异，已行移植的１３５例受者分为配型选择组５１例和随机选择组８４例，配型组０～１个位上错配率５４．９％，明显高于随机组的２８     

HLA配型不合的骨髓加外周血干细胞移植治疗重型再生障碍性贫血

再生障碍性贫血（AA）是临床常见的难治性血液疾病，尤其是重型再生障碍性贫血（SAA），病情凶险，1年死亡率达91％。随着异基因造血干细胞移植（allo-HSCT）用于SAA的治疗，SAA的长期生存率得到明显提高，可达到60％～90％。我们为1例SAA患者进行了亲缘性人类白细胞抗原（HLA）1个位点不合的骨髓加外周血干细胞移植获得成功，报道如下。     

血缘供者HLA一个位点不合的造血干细胞移植九例

异基因造血干细胞移植（allo-HSCT）是血液疾病的重要治疗方法之一。目前allo-HSCT中造血干细胞的主要来源是人类白细胞抗原（HLA）相合的血缘同胞供者，但在同胞中找到的比例仅25％，限制了其广泛应用。2003年4月至2006年9月，我们对9例血液病患者尝试了采用HLA一个位点不合的亲属作为供者，进行allo—HSCT，总结报道如下。     

肾移植供受者HLA－DR基因配型结果分析（附276例报告）

为了进一步提高肾移植的水平，采用顺序特异引物聚合酶链反应（ＰＣＲ－ＳＳＰ）方法用于２７６例肾移植供受者ＨＬＡ－ＤＲ基因配型，以探讨其临床可行性和实用性．结果显示：２７６例供受者３０８份样本，ＰＣＲ－ＳＳＰ分型均获成功，检测时间５小时内。供受者ＤＲ等位基因频率分布大致相同，无显著性差异。已行移植的１３５例受者分为配型选择组５１例和随机选择组８４例。配型组０～１个位点错配率５４．９％，明显高于随机组的２８．６％，而２个位点错配率为４５．１％，明显低于随机组的７１．４％，具有非常显著性差异（Ｐ＜０．０１）。研究表明，ＰＣＲ－ＳＳＰ快速ＨＬＡ－ＤＲ基因分型技术选择供受者适合于临床应用并具有很强的实用性。     

抗HLA和抗MICA抗体的表达对移植肾功能和急性排斥反应的预示作用

目的 探讨抗HLA与抗主要组织相容性复合物Ⅰ类链相关基因A(MICA)抗体的表达对移植肾功能和急性排斥反应的预示作用.方法 采用免疫磁珠流式液相芯片技术检测41例肾移植受者移植前后的抗HLA和抗MICA抗体,其中37例接受了1、3、6个月及1、2、3年的动态随访.分析抗HLA和抗MICA抗体的特异性,及其与血清肌酐和排斥反应的相关性.结果 移植前共有9例(22.0%,9/41)预存抗HLA或(和)抗MICA抗体,其巾抗HLA抗体阳性2例(4.9%,2/41),抗MICA抗体阳性6例(14.6%,6/41),抗HLA和抗MICA抗体均阳性1例(2.4%,1/41).另外有5例抗MICA抗体可疑阳性.除1例的抗MICA抗体为供者特异性抗体(DSA)外,其余均为非供者特异性抗体(NDSA).37例随访者中,6例新生抗HLA抗体(16.2%),3例新生抗MICA抗体(8.1%),新生抗HLA抗体者的抗体滴度在随访3年中呈现上升趋势.9例预存抗体的受者,有4例(44.4%,4/9)发生排斥反应;6例新生抗HLA抗体的受者中,有3例(50.0%)发生急性排斥反应,而3例新生抗MICA抗体的受者均无排斥反应发生,二者间的差异有统计学意义(P＜0.05).新生抗HLA抗体产生较早(术后3 d和7 d)的2例受者均检测到抗HLAⅡ类DSA,其移植肾均因排斥反应而切除.预存抗MICA抗体,且移植后发生排斥反应者在随访的每个时间点上的血清肌酐水平均高于预存抗MICA抗体但无排斥反应者(P＜0.05);移植前抗HLA和抗MICA抗体均阴性者,术后发生排斥反应者在随访的每个时间点上的血清肌酐水平均高于抗体阴性且无排斥反应者(P＜0.01);无论是新生抗HLA抗体还是抗MICA抗体,移植后1个月发生排斥反应者的血肌酐均明显高于抗体阴性且无排斥反应者(P＜0.01).结论 预存和新生抗HLA抗体是移植后发生急性排斥反应的重要因素,而供、受者HLA和MICA基因错配是产生新生抗体的重要原因.     

HLA基因与乙肝相关性肾炎相关性的研究综述

人类白细胞抗原（HLA）是最早发现的与疾病有明确关系的遗传系统,具有多态性、多基因性及高度特异性。作为调节机体免疫应答的重要基因群,它与疾病的免疫应答密切相关,并在抗原识别、提呈与调控等方面起重要作用。乙型肝炎病毒（HBV）为DNA病毒,感染机体后先合成前基因组mRNA,进而通过反转录合成完整的HBVDNAE。肾脏是HBV感染后常累及的器官,通过炎性反应、补体激活及电荷中和等造成肾小球基底膜和足细胞的损伤,     

抗HLA和抗MICA抗体的表达对移植肾功能和急性排斥反应的预示作用

Objective To investigate the prediction of anti-human leukocyte antigen antibodies (HLA) and anti-major histocompatibility complex class I-related chain A antibodies (MICA) to the development of acute rejection (AR) and kidney allograft function. Methods Forty-one kidney transplant patients were prospectively tested for anti-HLA and anti-MICA. Thirty-seven patients were screened using Luminex/single-antigen beads to determine the HLA and MICA-specific antibody levels at 0,30,90, 180,360,720 and 1080 days post-transplantation. The patients and donors of HLA and MICA allele typing were determined by PCR-SSOP, and donor specific antibody (DSA) and non-donor specific antibody (NDSA) were identified.Simultaneously,their serum creatinine (SCr) levels and clinical data were analyzed. Results Nine patients (21.95 % ,9/41 ) had pre-existing anti-HLA and(or) anti-MICA, including 6 cases of anti-MICA,2 cases of anti-HLA, and one case of anti-MICA and anti-HLA. Nine patients had pre-existing DSA and NDSA. In the 37 patients, 6 patients (16.2% ) developed de novo anti-HLA, and 3 (8.1%) developed de novo antiMICA. In patients positive for de novo anti-HLA, the titer of antibody was gradually increased during the follow-up of three years. Four patients out of 9 patients with pre-existing antibodies were suffered from AR (44.4%); In 6 patients positive for de novo anti-HLA,three cases (50.0%) were suffered from AR; In three patients positive for de novo anti-MICA,no AR occurred (P＜0.05). In two patients positive for DSA of HLAⅡ antibody detected at the third and seventh day after transplantation, the renal grafts were renovecd due to rejection. The Scr levels in patients positive for pre-existing MICA with AR were higher than in those positive for pre-existing MICA without AR at each scheduled time point during the follow-up period (P＜0.05). The Scr levels in patients negative for antibodies pre-transplantation and having AR were higher than in those having no AR at each scheduled time point during the follow-up period (P＜0. 01 ). The Scr levels in patients positive for de novo HLA and MICA and having AR one month following transplantation were higher than in those negative for antibodies and having no AR (P＜0.01 ). Conclusion Pre-existing and de novo anti-HLA were the irnportant factors for the development of AR, but the mismatch of HLA and MICA alleles in donors and patients was primary causes for generation of de novo antibodies.     

抗HLA和抗MICA抗体的表达对移植肾功能和急性排斥反应的预示作用

Objective To investigate the prediction of anti-human leukocyte antigen antibodies (HLA) and anti-major histocompatibility complex class I-related chain A antibodies (MICA) to the development of acute rejection (AR) and kidney allograft function. Methods Forty-one kidney transplant patients were prospectively tested for anti-HLA and anti-MICA. Thirty-seven patients were screened using Luminex/single-antigen beads to determine the HLA and MICA-specific antibody levels at 0,30,90, 180,360,720 and 1080 days post-transplantation. The patients and donors of HLA and MICA allele typing were determined by PCR-SSOP, and donor specific antibody (DSA) and non-donor specific antibody (NDSA) were identified.Simultaneously,their serum creatinine (SCr) levels and clinical data were analyzed. Results Nine patients (21.95 % ,9/41 ) had pre-existing anti-HLA and(or) anti-MICA, including 6 cases of anti-MICA,2 cases of anti-HLA, and one case of anti-MICA and anti-HLA. Nine patients had pre-existing DSA and NDSA. In the 37 patients, 6 patients (16.2% ) developed de novo anti-HLA, and 3 (8.1%) developed de novo antiMICA. In patients positive for de novo anti-HLA, the titer of antibody was gradually increased during the follow-up of three years. Four patients out of 9 patients with pre-existing antibodies were suffered from AR (44.4%); In 6 patients positive for de novo anti-HLA,three cases (50.0%) were suffered from AR; In three patients positive for de novo anti-MICA,no AR occurred (P＜0.05). In two patients positive for DSA of HLAⅡ antibody detected at the third and seventh day after transplantation, the renal grafts were renovecd due to rejection. The Scr levels in patients positive for pre-existing MICA with AR were higher than in those positive for pre-existing MICA without AR at each scheduled time point during the follow-up period (P＜0.05). The Scr levels in patients negative for antibodies pre-transplantation and having AR were higher than in those having no AR at each scheduled time point during the follow-up period (P＜0. 01 ). The Scr levels in patients positive for de novo HLA and MICA and having AR one month following transplantation were higher than in those negative for antibodies and having no AR (P＜0.01 ). Conclusion Pre-existing and de novo anti-HLA were the irnportant factors for the development of AR, but the mismatch of HLA and MICA alleles in donors and patients was primary causes for generation of de novo antibodies.     

引导性骨再生模型（GBR）成骨特点及骨髓的作用

目的∶研究引导性骨再生模型（ＧＢＲ）的成骨特点及骨髓的作用。材料与方法∶本实验应用了２４只新西兰兔，兔右侧桡骨为ＧＢＲ侧，左侧为去骨髓侧。ＧＢＲ侧手术过程：于兔右侧桡骨做１０ｍｍ骨缺损，以硅胶管套于缺损两侧；去骨髓侧以骨水泥封堵缺损两端髓腔。动物于３日、１、２、３、４、６、１０、１２周处死，标本做非脱钙骨切片，分析大体观察、Ｘ线及非脱钙骨组织切片结果。结果：（１）骨髓在ＧＢＲ模型膜管内成骨过程中起决定作用；（２）ＧＢＲ模型骨修复方式：于膜管内骨缺损两端分别形成两成骨尖端，两成骨尖端相对生长至愈合；（３）膜管内成骨过程早期为成骨细胞直接成骨过程，后期成骨尖端缺乏成熟皮质骨结构。结论：ＧＢＲ模型基础为隔膜干扰技术，其骨缺损修复为一特殊成骨过程，骨髓对其起决定作用。     

骨髓基质细胞体外培养骨发生潜能及条件研究

目的：建立一种骨髓基质细胞（ＢＭＳｃ）向成骨细胞转化的体外培养方法,观察其成骨过程,并探讨部分因子在ＢＭＳｃ转化及增殖中的作用。方法：将兔ＢＭＳｃ悬液进行体外培养,进行形态学观察和组织学检测;检测ｂＦＧＦ对ＢＭ－Ｓｃ的促增殖作用。结果：传代培养的细胞ＡＬＰ阳性率达８０％以上,细胞逐渐分化散在致密的岛状结构并分泌细胞外基质形成钙结节;ｂＦＧＦ能刺激ＢＭＳｃ的增殖。结论：培养的ＢＭＳｃ在体外仍具有成骨     

可注射型生物蛋白胶包埋骨髓基质细胞工程化组织的体外实验

目的构建可注射型生物蛋白胶包埋骨髓基质细胞的工程化组织,体外培养并研究其生物学特性,探讨将可注射型生物蛋白胶作为组织工程支架用于临床的实验基础。方法体外培养浇铸有骨髓基质细胞的生物蛋白胶,通过倒置相差显微镜、激光共聚焦显微镜观察载体内细胞生长及载体降解情况,5-溴脱氧尿苷(5-Bromodeocyuridine,BrdU)掺入标记后免疫组化等方法研究可注射型载体内包埋细胞的增殖情况。结果骨髓基质细胞包埋于生物蛋白胶内能很好地存活并增殖,2d后细胞呈典型的成纤维细胞形态;6d后生物蛋白胶边缘部分开始降解,细胞脱落至培养板;体外培养14d,细胞生长良好,大部分生物胶降解,脱落的细胞增多,贴壁生长的细胞形态正常;3周后生物蛋白胶完全降解。结论生物蛋白胶聚合后包埋的种子细胞能够正常增殖,生物蛋白胶是一种理想的适用于微创方法修复组织的可注射型组织工程培养和移植的支架。     

骨基质明胶复合自体红骨髓及同种异体骨修复骨缺损

目的:评价骨基质明胶复合自体红骨髓及同种异体骨联合移植修复骨缺损的疗效。方法:76例良性骨肿瘤和瘤样病损患者,彻底刮除病灶或作肿瘤骨段切除,并对瘤壁作灭活处理,以同种异体骨作支架,周围填充骨基质明胶和自体红骨髓复合物,术后观察机体反应及骨缺损修复情况。结果:术后机体无明显免疫排斥反应,无1例发生感染,所有病例随访时间为5～16个月,X线显示新骨形成时间为术后1.5～4月,完全骨化的时间为术后5～9月,骨缺损骨性愈合74例,并获得较好的关节功能,肿瘤复发2例。结论:骨基质明胶、自体红骨髓、同种异体骨复合物能有效修复骨缺损,有广泛的临床应用前景。     

Normal Donor Bone Marrow is Superior to Flt3 Ligand-Mobilized Bone Marrow in Prolonging Heart Allograft Survival when Combined with Anti-CD40L (CD154)

Flt3 ligand (FL) administration markedly increases bone marrow (BM) stem cells and immature dendritic cells. We investigated the influence of CD40-CD40Ligand (CD154) pathway blockade on antidonor immunity, cytokine production, microchimerism and heart graft survival in BALB/c (H2d) recipients of fully allogeneic C57BL/10 (H2b) FL-mobilized BM (FL-BM) or normal BM. Anti-CD40L mAb strongly suppressed anti-donor T-cell proliferative responses in recipients of either normal or FL-BM, but was less efficient in inhibiting antidonor cytolytic T-cell (CTL) activity, especially in recipients of FL-BM. Interestingly, CD40L blockade was more effective in recipients of multiple compared with single donor BM infusions. Anti-donor cytokine responses revealed complete impairment of IFN-gamma, IL-4 and IL-10 production in recipients of normal BM and CD40L mAb. By contrast, and in agreement with the CTL data, mice given FL-BM retained ability to produce IFN-gamma CD40-CD40L blockade did not promote microchimerism, as evidenced by immunohistology and real time polymerase chain reaction. Nevertheless, anti-CD40L mAb enhanced heart allograft survival in recipients of FL-BM, but the effect was inferior to that achieved with normal BM. These data provide insight into the influence of growth factor-expanded donor BM and costimulation blockade on antidonor immune reactivity and transplant outcome. The comparatively poor outcome obtained using FL-BM plus anti-CD40L mAb in this model may be ascribed to the failure of effectively interdicting antidonor CTL activity.     

壳聚糖海藻酸钠微球与骨髓基质细胞相容性实验研究

目的：研究壳聚糖海藻酸钠复合微球的生物相容性。方法：将壳聚糖与海藻酸钠制成复合微球,采用体外细胞培养技术,将微球与细胞一起培养,通过对细胞增殖率及ALP活性、细胞微量蛋白的检测,观察微球对培养细胞的影响。结果：壳聚糖海藻酸钠复合微球对骨髓基质细胞的增殖、分化及分泌功能均无明显影响。结论：壳聚糖海藻酸钠微球生物相容性良好。     

Mechanisms of Alloimmunization and Subsequent Bone Marrow Transplantation Rejection Induced by Platelet Transfusion in a Murine Model

For many nonmalignant hematological disorders, HLA‐matched bone marrow transplantation (BMT) is curative. However, due to lack of neoplasia, the toxicity of stringent conditioning regimens is difficult to justify, and reduced intensity conditioning is used. Unfortunately, current reduced intensity regimens have high rates of BMT rejection. We have recently reported in a murine model that mHAs on transfused platelet products induce subsequent BMT rejection. Most nonmalignant hematological disorders require transfusion support prior to BMT and the rate of BMT rejection in humans correlates with the number of transfusions given. Herein, we perform a mechanistic analysis of platelet transfusion‐induced BMT rejection and report that unlike exposure to alloantigens during transplantation, platelet transfusion primes alloimmunity but does not stimulate full effector function. Subsequent BMT is itself an additional and distinct immunizing event, which does not induce rejection without antecedent priming from transfusion. Both CD4⁺ and CD8⁺ T cells are required for priming during platelet transfusion, but only CD8⁺ T cells are required for BMT rejection. In neither case are antibodies required for rejection to occur .     

犬骨髓单个核细胞构建组织工程骨的体外实验研究

目的 探索组织工程骨(Tissue Engineered Bone,TEB)的即刻构建方案,以符合临床骨缺损即刻修复的要求。方法 分离获取Beagle犬骨髓单个核细胞(Canine bone marrow mononuclear cells,cBMNCs)。将密度为30×10~6 cells/mL的cBMNCs复合部分脱钙骨(Partially demineralized bone matrix,pDBM)即刻构建TEB。对即刻构建的TEB进行体外培养和成骨检测。结果 在成骨诱导的体外培养环境下,即刻构建的TEB具有一定的细胞活性和成骨能力。结论 即刻构建的TEB可直接回植,以满足体内即刻骨缺损修复的要求。