A chimeric tyrosine/tryptophan hydroxylase

The neurotransmitter biosynthetic enzymes, tyrosine hydroxylase (TH), and tryptophan hydroxylase (TPH) are each composed of an amino-terminal regulatory domain and a carboxylterminal catalytic domain. A chimeric hydroxylase was generated by coupling the regulatory domain of TH (TH-R) to the catalytic domain of TPH (TPH-C) and expressing the recombinant enzyme in bacteria. The chimeric junction was created at proline 165 in TH and proline 106 in TPH because this residue is within a conserved five amino-acid span (ValProTrpPhePro) that defines the beginning of the highly homologous catalytic domains of TH and TPH. Radioenzymatic activity assays demonstrated that the TH-R/TPH-C chimera hydroxylates tryptophan, but not tyrosine. Therefore, the regulatory domain does not confer substrate specificity. Although the TH-R/TPH-C enzyme did serve as a substrate for protein kinase (PKA), activation was not observed following phosphorylation. Phosphorylation studies in combination with kinetic data provided evidence that TH-R does not exert a dominant influence on TPH-C. Stability assays revealed that, whereas TH exhibited a t_1/2 of 84 min at 37°C, TPH was much less stable (t _1/2=28.3 min). The stability profile of TH-R/TPH-C, however, was superimposable on that of TH. Removal of the regulatory domain (a deletion of 165 amino acids from the N-terminus) of TH rendered the catalytic domain highly unstable, as demonstrated by at _1/2 of 14 min. The authors conclude that the regulatory domain of TH functions as a stabilizer of enzyme activity. As a corollary, the well-characterized instability of TPH may be attributed to the inability of its regulatory domain to stabilize the catalytic domain.     

子-母微量嵌合体基础上同胞间非T淋巴细胞去除HLA半相合造血干细胞移植一例

目的为扩大供者来源,探讨在子-母微量嵌合体基础上同胞间非T淋巴细胞去除(Non-TCD)HLA半相合造血干细胞移植的可行性。方法受者的原发病为慢性粒细胞白血病(CML)急性淋巴细胞病变,供者为其胞弟,供、受者HLA有3个抗原不同,经套式序列特异引物聚合酶链反应技术检测,供者微量嵌合体阳性。采用全身照射、司莫司汀、阿糖胞苷、环磷酰胺及兔源抗胸腺细胞球蛋白等对受者进行预处理;采用环孢素A、霉酚酸酯及甲氨蝶呤预防移植物抗宿主病(GVHD)。结果移植后受者的外周血中性粒细胞>0.5×109/L和血小板>20×109/L的时间分别为11、18d,骨髓检查显示增生活跃,粒细胞系、红细胞系形态和比例正常;1、2、3、6个月和1年时完全供者型嵌合>90%。术后发生Ⅱ度急性GVHD及慢性局限性GVHD,经调整免疫抑制治疗方案后缓解。受者现基本恢复正常生活。结论子-母微嵌合体阳性的HLA半相合同胞可作为Non-TCD造血干细胞移植的供者。     

人HLA半相合混合脐血在SCID小鼠移植的实验研究

目的：探讨不同供者来源脐血混合移植的可行性和植入特性。方法：分别将两份人HLA半相合混合脐血或单份脐血输入经亚致量照射后的严重联合免疫缺陷（SCID）小鼠，观察两组脐血在SCID小鼠体内的植入状况及多系造血重建特性。结果：混合脐血和单份脐血移植均可在受鼠体内植入，形成供－受混合嵌合体，并能重建多系造血，存活率和植入率无统计学意义（P＞0．05）。用多聚酶链反应－序列特异性寡核苷酸（PCR－SSO）探针检测人HLA－DQB1基因发现，HLA半相合混合脐血移植可有1份或2份脐血植入，其中造血祖细胞含量和体外克隆形成能力高者，更易于植入，造血重建特性与单一脐血移植比较无统计学意义。结论：HLA半相合人混合脐血可同时在SCID小鼠体内植入，形成来自供－受三者的多嵌合状态，并能重建造血及免疫功能。     

胚胎植入前遗传学筛查的临床应用

随着辅助生殖技术和遗传学分析技术的发展,胚胎植入前遗传学筛查应用于胚胎染色体数目异常（非整倍体）检测,以期改善体外受精-胚胎移植的妊娠结局。新方法、新技术不断出现并应用于胚胎植入前遗传学筛查中,如囊胚期活检、比较基因组杂交技术、微阵列技术、第二代测序技术等,显著增加了诊断准确性,减少误诊风险。同时,胚胎植入前遗传学筛查的广泛应用也面临许多挑战。综述该领域的应用进展和面临的挑战。     

骨髓移植诱导临床心脏移植后供者特异性免疫耐受方案的探讨

目的 探讨骨髓移植诱导临床心脏移植后供者特异性免疫耐受的可行性.方法 采取供心的同时采用改良"灌流法"获取供者的骨髓350 ml,经过滤及离心处理后,加入细胞冷冻保护液共80ml,分装于低温冻存袋,经程序降温,置于-80℃冰箱中保存.在常规原位心脏移植术后40 d,取冻存骨髓快速复温,穿刺受者双侧髂后上嵴,立即行骨髓腔内骨髓细胞输注(IBM-BMT),共输注单核细胞1.2×107/kg,CD34+细胞2.38×105/kg.骨髓输注前3 d行预处理,包括应用氟达拉滨、抗胸腺细胞球蛋白及全身淋巴结照射.骨髓移植后静脉应用他克莫司(Tac),维持血Tac浓度谷值在10～20μg/L;3周后改为口服Tac+吗替麦考酚酯(MMF);6周后改为环孢素A及MMF.分别于心脏移植后2、4、8和12周采集受者外周血,分别于术后4、8和12周采集受者的骨髓,应用短串联重复序列-聚合酶链反应法检测供者嵌合体.心脏移植后每周行心肌内心电图检查,每月行心肌活检1次.术后3个月,取受者及第三者外周血单核细胞,行混合淋巴细胞反应(MLR).结果心脏移植后1、2及3个月时受者的外周血及髂骨内骨髓细胞中供者来源的细胞比例分别为26.3%、19.1%、4.8%和46.3%、24.4%、7.6%.IBM-BMT后心肌内心电图监测显示心肌阻抗及R波波幅无明显变化.术后3个月行心内膜心肌活检,未见排斥反应征象.术后3个月时行超声心动图检查,提示心脏舒张、收缩功能良好.MLR提示受者对供者特异性刺激呈现低反应性,而对第三者仍保持良好的免疫活性(P＜0.01).结论 采取分期骨髓移植免疫耐受诱导方案可安全、有效地建立嵌合体,成功诱导心脏移植后供者特异性免疫耐受,但远期效果有待进一步研究.

Abstract：

Objective To investigate a new strategy of bone marrow transplantation (BMT) for donor-specific tolerance induction after heart transplantation. Methods Donor bone marrow cells (BMCs)were harvested simultaneously with donor cardiac graft using modified perfusion method (PM) ,then stored in a -80 ℃ refrigerator after filtration and centrifugation. Whole BMCs (IBM-BMT) (monocytes 1.2 ×107/kg,CD34+ cells 2.38× 105/kg) in host iliac bones were injected into the bone marrow cavity 40 days after heart transplantation. Preconditoning regimens that consisted of fludarabine, antithymoctye globin and total lymphoid irradiation were performed 3 days before BMT. Tacrolimus (Tac) was administrated intravenously after BMT or orally in conjunction with mycophenolate mofetil (MMF) 3 weeks later.Cyclosporine and MMF were orally administrated 6 weeks later. Donor chimerism was detected using short tandem repeats-polymerase chain reaction in monocytes from peripheral blood at the 2nd,4th, 8th or 12th week after BMT or BMCs at the 4th, 8th or 12th week after BMT. Intramyocardium electrocardiography examination or endomyocardial biopsy was performed weekly or monthly respectively. Mixed lymphocyte reactions (MLR) were performed 3 months after BMT. Results Donor chimerism in monocytes in peripheral blood or BMCs in iliac bones measured at the 1 st,2nd and 3rd month after BMT was 26.3%, 19.1%,4.8% ,and 46.3%, 24.4%, 7.6%, respectively. After 3-month follow-up, there was no rejection confirmed by endomyocardial biopsy or intramyocardium electrocardiography. Echocardiography revealed that the diastolic and systolic function of the cardiac graft was maintained well 3 months after BMT. MLR revealed donor-specific hyporesponsiveness while immunocompetence was preserved to third-party antigens. Conclusion These findings indicate that the two-stage BMT strategy is a safe and feasible method for the induction of donor-specific tolerance via stable mixed chimerism and needs to be further confirmed after a long-term observation.     

A tymovirus with an atypical 3′-UTR illuminates the possibilities for 3′-UTR evolution

We report the complete genome sequence of Dulcamara mottle virus (DuMV), confirming its membership within the Tymovirus genus, which was previously based on physical and pathology evidence. The 5′-untranslated region (UTR) and coding region of DuMV RNA have the typical characteristics of tymoviral RNAs. In contrast, the 3′-UTR is the longest and most unusual yet reported for a tymovirus, possessing an internal poly(A) tract, lacking a 3′-tRNA-like structure (TLS) and terminating at the 3′-end with –UUC instead of the typical –CC(A). An expressible cDNA clone was constructed and shown to be capable of producing infectious DuMV genomic RNAs with –UUC 3′-termini. A chimeric Turnip yellow mosaic virus (TYMV) genome bearing the DuMV 3′-UTR in place of the normal TLS was constructed in order to investigate the ability of the TYMV replication proteins to amplify RNAs with –UUC instead of –CC(A) 3′-termini. The chimeric genome was shown to be capable of replication and systemic spread in plants, although amplification was very limited. These experiments suggest the way in which DuMV may have evolved from a typical tymovirus, and illuminate the ways in which viral 3′-UTRs in general can evolve.     

成年大鼠联体诱导的移植耐受与嵌合状态相关性的研究

目的 将异基因成年大鼠联体共生（ｐａｒａｂｉｏｓｉｓ）以诱导相互特异的移植耐受，探讨嵌合体与耐受的关系。方法 联体前后低剂量环磷酰胺（ＣＰ，１００ｍｇ／ｋｇ）腹腔注射，观察联体大鼠存活时间。分别于联体５、１５、４５ｄ时将联体大鼠分开，并相互植皮，观察皮肤存活情况，并在上述时间点以流式细胞仪检测联体分开后大鼠胸腺、脾脏中供体来源细胞的嵌合状态。在联体１５ｄ时检测同种异型混合淋巴细胞反应（ＭＬＲ）、迟     

小鼠到大鼠骨髓移植诱导供者特异的免疫耐受

目的　探索适用临床的异种骨髓移植诱导免疫耐受的方案。方法　受体SD大鼠接受亚致死剂量照射(5 5Gy)当天尾静脉注射BALB/c小鼠骨髓细胞 8× 10 7,2d后腹腔内注射环磷酰胺 15 0mg/kg。分别于移植后 3 0 ,60及90d取受体大鼠外周血 ,用特异性单抗作直接免疫荧光染色后上流式细胞仪 (FACS)分析 ,检测骨髓植入的嵌合情况 ,并于 4周后作皮片移植和混合淋巴反应 (MLR) ,检查免疫耐受情况。结果　受体大鼠外周血可检测到小鼠骨髓源性细胞 ,嵌合体在 90d时仍存在。皮片移植和MLR证明受体SD大鼠对供体BALB/c小鼠产生特异性免疫耐受 ,对无关第 3者C5 7BL/ 6小鼠仍有强烈免疫应答。结论　小鼠到大鼠骨髓移植成功地诱导了特异性免疫耐受。