利用T细胞受体重排删除环和TCRBV克隆谱型研究异基因造血干细胞移植后T细胞免疫重建

目的通过T细胞受体重排删除环（TREC）和T细胞受体（TCRBV）克隆谱型研究异基因造血干细胞移植（allo—HSCT）后T细胞免疫重建。方法应用实时PCR的方法定量检测43例3组不同类型allo—HSCT后及70名正常供者TREC水平;应用RT—PCR扩增其中24例3组不同类型allo—HSCT后及5名正常供者的外周血的TCRBV24个家族的基因序列,利用基因扫描的方法判断TCRBV家族的克隆表达、互补决定区3（CDR3）克隆性质及计算BV家族的利用率。结果移植前患者TREC定量检测结果为（335．1±782．5）拷贝／10^5细胞,明显低于正常人。3组在移植后3个月TREC含量均明显下降,同胞相合骨髓移植（MSD—BMT）组TREC恢复快于其他两组,且在移植后24个月基本恢复到移植前水平。单倍型骨髓移植（HID—BMT）组的TREC恢复延迟。在移植后3—19个月内TCRBVCDR3克隆谱型仍处于不均一状态,在6～16个BV家族中有表达。多克隆表达率在33％～48％,其余为单克隆或寡克隆增生,且分布在24个不同的BV家族,未见共用的BV家族。结论通过TREC和TCRBV克隆谱型分析,移植后3～24个月,胸腺输出初始T细胞数量低水平及TCRBV家族克隆性利用。单倍型骨髓移植后T细胞免疫缺陷更严重,与临床经过一致。     

单倍型骨髓移植小鼠移植物抗宿主病模型的建立及移植物抗宿主病靶器官T细胞受体克隆检测

目的:建立单倍型骨髓移植小鼠模型,观察单倍型骨髓移植小鼠移植物抗宿主病(GVHD)靶器官的组织病理表现及器官特异性T细胞受体克隆表达.方法:建立单倍型异基因移植小鼠GVHD模型,进行移植前后GVHD靶器官的病理分析,同时应用RT-PCR扩增小鼠肝脏、皮肤、结肠、肾脏等组织移植前、后T细胞受体β链可变区(TCRBV)20个家族的基因序列,通过基因扫描判断TCRBV家族的克隆表达和CDR3克隆的性质.结果:单倍型骨髓移植小鼠在移植后14 d临床开始出现典型的GVHD表现.移植后24 d受鼠肝脏、皮肤、远端回肠等均出现典型的GVHD病理表现,在其T细胞受体谱型图上出现了分属TCRBV家族的单克隆或寡克隆增生的T细胞,而肾脏等非GVHD靶器官中浸润的淋巴细胞为多克隆的T细胞增生.结论:单倍型骨髓移植小鼠在移植后14 d出现典型的GVHD表现,GVHD靶器官出现的T细胞单克隆及寡克隆增生可能与GVHD的发生相关.     

外周造血干细胞移植后及出现GVHD时T细胞β链CDR3谱型分析

目的 初步探讨外周造血干细胞(PBSC)移植后以及出现移植物抗宿主疾病(GVHD)时,外周血T细胞a链的CDR3谱型变化和特征.方法 采用免疫扫描谱型分析技术,监测1例重型β-地中海贫血移植前、移植后23 d、GVHD时(移植后28 d)患者外周血单核细胞(PBMC)及供者PBSC标本T细胞a链的CDR3谱型变化,基因扫描(GeneScan)和测序分析克隆性增生T细胞TCR分子特征.结果 患者移植前PBMC的24个TCR BV家族CDR3谱型均呈高斯分布,部分家族在移植后23 d、和GVHD时呈现为寡峰和单峰,同时部分家族消失,单克隆增生T细胞TCR beta链的测序结果 显示不同的CDR3区组成,设计特异的CDR3引物进一步分析提示其可能来源于干细胞移植后的分化.结论 PBSC移植后23 d,PBMC中的多数24TCRBV家族CDR3谱型表现为多克隆,GVHD时,部分家族出现明显的单、寡克隆增生,这些特异T细胞可能在GVHD中发挥重要作用.对移植前后TCRCDR3谱型和特异TCR分子特征的分析将有助于PBSC移植后免疫重建的评估和出现排斥反应时的特异性治疗.     

慢性特发性血小板减少性紫癜相关T细胞克隆的T细胞受体特征

目的研究与特发性血小板减少性紫癜（ITP）发病相关的反应性T细胞克隆的T细胞受体13链可变区（TCRBV）基因特征。方法应用逆转录一聚合酶链反应（RT—PCR）扩增25例ITP患者和20名正常人外周血以及20例脐带血的TCRBV24个家族的基因序列,在长泳道测序胶上电泳,形成TCRBV互补决定区3（CDR3）基因表达谱型图。通过谱型图上电泳条带分析TCRBV基因分布特征,切割代表性条带测序。结合临床特点进行分析。结果（1）急性ITP（aITP）和慢性ITP（cITP）的TCRBV CDR3基因表达谱型图有明显不同,15例aITP浓集条带与正常对照组相比,差异无统计学意义（P=0．179）。10例cITP则都出现以一些浓集条带为代表的寡克隆T细胞增生,浓集条带明显多于正常对照组（P〈0．05）。其中6例cITP在TCRBV8亚家族分别出现1～2条浓集条带,共8条浓集条带,3例cITP在TCRBV 13．1亚家族各出现1条浓集条带,4例cITP在TCRBV 14亚家族出现5条浓集条带,3例cITP在TCRBV17亚家族出现4条浓集条带。将这20个浓集条带直接测序,有19个条带测序为单克隆T细胞扩增,1条BV14条带测序呈多克隆性。（2）比较10例cITP患者T细胞克隆的TCRBV基因或者编码CDR3的氨基酸,其中2例患者的TCRBV8全部基因序列相同,另有3例患者的TCRBV13．1全部基因序列相同。4例cITP患者中有2例的T细胞克隆TCRBV17的CDR3完全一致,全部序列仅仅框架区4有3个氨基酸不同,另有2例的T细胞克隆的TCRBV17的CDR3序列仅有4个氨基酸不同,表明不同cITP患者有同样或者功能类似的TCRBV基因编码的T细胞克隆增殖。不同患者分属不同TCRBV基因家族的19个T细胞克隆的CDR3区共用3种模体,有7个T细胞克隆的TCRBV CDR3共用模体E（D）TQYFGPG;4个T细胞克隆的TCRBV CDR3共用模体N（K）EQFFGPG;4个TCRBV17的T细胞克隆中有3个T细胞克隆的TCRBV17 CDR3共用模体GANVLTTGAG。结论cITP发病与特定的T细胞寡克隆扩增有关,这些T细胞克隆的CDR3共享3种可能结合同样抗原的模体。aITP没有明显的T细胞克隆变化。     

异基因造血干细胞移植治疗急性髓系白血病4例效果分析

目的：回顾性分析急性髓系白血病异基因造血干细胞移植的临床疗效。方法：急性髓系白血病患者4例,采用粒细胞集落刺激因子动员外周血干细胞,其中3例行全相合异基因移植,1例行半相合异基因移植。结果：4例患者均获得造血重建,呈完全嵌合体状态,粒细胞中位植活时间为11（11～12）ｄ,血小板中位植活时间为12.5（11～13）ｄ。2例出现aGVHD,其中Ⅰ度1例,Ⅳ度1例;2例出现cGVHD,1例为局限性,1例为Ⅳ度肠、肝cGVHD;1例出现消化道粘膜炎,1例出现轻度肝功能损害,2例EB病毒定量升高,治疗后均好转。结论：异基因造血干细胞移植治疗AML疗效显著,使用外周血干细胞进行半相合移植值得推广。     

异基因外周血造血干细胞移植后细胞免疫的重建

目的 研究异基因外周血造血干细胞移植后细胞免疫的重建，探讨免疫治疗应用于造血干细胞移植后的残留病灶清除，并将其与化疗有机结合，以延长患者无病生存率的方法。方法 采用流式细胞仪，检测14例异基因外周血造血干细胞移植患者移植前后外周血中T、B、NK细胞的含量。结果 异基因外周血造血干细胞移植后1月内，患者血循环中CD3^＋细胞明显减少，至移植后3月逐渐恢复至正常；CD3^＋CD4^＋细胞移植后持续减少，至移植后6月甚至更长时间后才逐渐恢复；CD3^＋CD8^＋细胞于移植后1月内很快恢复。CD19^＋细胞于移植后l～3月降低，移植后3～6月逐渐恢复。NK细胞于移植后2月恢复，较T或B淋巴细胞恢复速度快。结论 异基因外周血造血干细胞移植后3月T细胞才逐渐恢复至正常水平；B细胞于移植后1～3月绝对及相对计数降低，移植后3～6月逐渐恢复；NK细胞于移植后2月恢复，较T或B淋巴细胞恢复速度快。     

监测FasL表达评估异基因造血干细胞移植后T细胞免疫功能状态

目的评估异基因造血干细胞移植后Ｔ淋巴细胞免疫功能状态。方法采用三色荧光标记的流式细胞术,对４例异基因造血干细胞移植、２例自体干细胞移植患者移植前后的Ｔ淋巴细胞及其亚群的ＦａｓＬ表达进行监测。结果移植前Ｔ细胞的ＴａｓＬ、表达率仅（２.１３％±２.０２％）,与正常对照组无显著差异（Ｐ＞０.０５）,移植后异基因造血干细胞移植患者ＦａｓＬ表达水平升高,尤以急性ＧＶＨＤ患者表达升高显著（Ｐ<０.０５）,而自体干细胞移植患者的ＦａｓＬ表达未见明显改变。结论异基因造血干细胞移植术后部分Ｔ淋巴细胞处于激活状态; ＦａｓＬ表达可能与ａＧＶＨＤ相关;对ＦａｓＬ表达的监测可从细胞、免疫分子两个层次对Ｔ细胞免疫功能状态进行评估,该体系的完善对临床判断预后、指导治疗可能有较直接的参考价值。     

异基因外周血造血干细胞移植后细胞免疫的重建

目的 研究异基因外周血造血干细胞移植后细胞免疫的重建,探讨免疫治疗应用于造血干细胞移植后的残留病灶清除,并将其与化疗有机结合,以延长患者无病生存率的方法.方法 采用流式细胞仪,检测14例异基因外周血造血干细胞移植患者移植前后外周血中T、B、NK细胞的含量.结果 异基因外周血造血干细胞移植后1月内,患者血循环中CD3+细胞明显减少,至移植后3月逐渐恢复至正常;CD3+CD4+细胞移植后持续减少,至移植后6月甚至更长时间后才逐渐恢复;CD3+CD8+细胞于移植后1月内很快恢复.CD19+细胞于移植后1 ～ 3月降低,移植后3 ～ 6月逐渐恢复.NK细胞于移植后2月恢复,较T或B淋巴细胞恢复速度快.结论 异基因外周血造血干细胞移植后3月T细胞才逐渐恢复至正常水平;B细胞于移植后1 ～ 3月绝对及相对计数降低,移植后3 ～ 6月逐渐恢复;NK细胞于移植后2月恢复,较T或B淋巴细胞恢复速度快.     

异基因干细胞移植后T细胞重建及与慢性移植物抗宿主病关系的研究

目的:探讨恶性血液病异基因干细胞移植后T细胞免疫重建的特点及与慢性移植物抗宿主病(cGVHD)的关系.方法:采用流式细胞仪动态检测42例恶性血液病患者异基因干细胞移植前、后不同时期T淋巴细胞亚群的变化,并对其中18例白血病患者检测其移植前、后不同时期的CD3 CD5 、CD3 CD45RA 、CD4 CD45RA 细胞的变化情况.结果:42例患者均重建造血.移植后CD4 T细胞恢复较慢,而CD8 T细胞则恢复较快,导致CD4 /CD8 细胞比值在移植后1年内持续倒置.回输CD 34细胞数量分别与移植后30 d时的CD3 、CD4 、CD8 T细胞数呈正相关.cGVHD组患者的CD3 CD45RA 、CD4 CD45RA 细胞均明显低于无GVHD组患者(均P<0.05).结论:异基因干细胞移植后CD4 T细胞恢复慢,而CD8 T细胞恢复较快.回输CD 34细胞数与移植后早期患者T细胞重建的快慢密切相关.cGVHD可引起初始T细胞亚群(CD45RA 细胞)减少.     

造血干细胞移植患者动态监测胸腺输出功能的意义

目的观察造血干细胞移植患者胸腺近期输出功能的动态变化情况,分析其相关影响因素,从而了解移植后T细胞免疫重建情况。方法选择2007年1月~2012年6月于北京大学深圳医院接受造血干细胞移植的血液病患者41例,采用实时荧光定量PCR方法检测患者不同时期200μL外周血中T细胞受体重排DNA删除环（sjTREC）水平,计算每毫升血液中sjTREC拷贝数。同时检测32例年龄匹配的正常者外周血T细胞受体重排DNA删除环含量作为正常对照。比较患者造血干细胞移植后sjTREC随时间变化情况。结果 32例正常对照组外周血Log sjTREC/mL为3.78±0.22,它与年龄呈负相关（r=-0.59,P〈0.05）。41例移植患者移植前外周血sjTREC水平显著低于正常对照组,移植后外周血sjTREC进一步下降,移植6个月后TRECs逐渐升高,移植后1.5年基本恢复至移植前水平,移植后2.5年恢复至接近正常水平。反复化疗可使患者胸腺输出功能下降。不同年龄组患者移植后胸腺输出功能恢复差异无统计学意义（P〉0.05）。移植6个月后,自体造血干细胞移植组患者外周血sjTREC水平恢复快于异基因移植组（P〈0.05）,至移植后2年二者差异无统计学意义（P〉0.05）。移植1年后存在广泛型cGVHD的患者其外周血sjTREC水平下降,低于无cGVHD者（P〈0.05）。结论造血干细胞移植后T细胞免疫重建缓慢,并受多种因素影响。胸腺输出功能可作为评价移植后免疫重建的有效指标。     

APPLICATION OF LORNOXICAM TO PATIENT-CONTROLLED ANALGESIA IN PATIENTS UNDERGOING ABDOMINAL SURGERIES

FOR anesthesiologis s ,treatingpostoperativepainhas alwaysbeen a problem.Althoughopioidshave been provedtobe effective,theirsideeffectscouldnotbeignored.With thedevelopmentofscienceand pharmacology,many drugs with aspectsof satisfactoryanalgesicefficacyand couldbe welltoleratedby patientshave been developed.And lornoxicamisone of them, which isa non-steroidalanti-inflammatorydrug (NSAID ), with analgesic, anti-infl-ammatory,andantipyreticproperties.Itseliminationhalf-time(3 to 5 hours) isle…     

Acupuncture Treatment of Vomiting

CASE HISTORY A female patient, 46 years old, head of the foreign affairs department of a certain university in Beijing, paid her first visit on October 9, 2006, with the chief complaint of vomiting for one month. She got vomiting after meals in early September. Before that, she had discomfortable sensation in the stomach due to angry with others, but she didn't pay much attention. Later, it developed into vomiting after eating. After the vomiting, the discomfort would be relieved, but with slight hypodynamia. She was once diagnosed as having 'neurogenic vomiting'. Having taken some western and Chinese drugs, the above symptoms were a little bit improved, but she would have nausea upon eating and with regurgitation. Because of the fear for vomiting, she did not dare to have food intake, with body weight reduction of 6 kilos in one month.     

Qiu Baoguo''''s Experience in Treating Consumptive Syndromes after Radio-Chemotherapies

Radiotherapy and chemotherapy are the important modern medical therapies for malignant tumors,yet they can also bring about serious local and systemic toxic side reactions so to decrease the patient;'s life quality,manifested by a series of consumptive symptoms.Having engaged in the combined work of Chinese and western medicine for nearly 50 years,the research fellow Qiu Baoguo in Henan Provincial Academy of TCM has developed his unique views on the TCM study of consumptive syndromes.The author of this essay had once the fortune tO follow Dr.Qiu in clinic,and specially would like to introduce in the following Dr.Qiu's experience in treating consumptive syndromes after radio-chemotherapies for patients with malignant tumor.     

Insomnia Due to Deficiency of Both the Heart and Spleen Treated by Acupuncture-Moxibustion and Chinese Tuina

OBJECTIVE: To observe therapeutic effects of the comprehensive therapy of acupuncture-moxibustion and Chinese Tuina for treatment of insomnia due to deficiency of both the heart and spleen. METHODS: 92 cases were divided randomly into the treatment group (treated by acupuncture-moxibustion and Chinese Tuina) and the control group (treated by acupuncture-moxibustion). RESULTS: The therapeutic effect of the treatment group was obviously superior to that of the control group (the CHI2 test showed P < 0.01). CONCLUSIONS: The comprehensive therapy of acupuncture-moxibustion and Chinese Tuina can give marked therapeutic effects for treatment of insomnia due to deficiency of both the heart and spleen.     

Analysis of CK-19,PDX-1,Nestin,Ngn3 in different donor islet purity in rats

Objective: To investigate if there are the CK-19, PDX-1, Nestin, Ngn3 positive cells in the donor islets of different purity in rats. Methods: Thirty male adult SD rats were randomly divided into 3 groups. Islets were isolated using digestion by ductal injection of collagenase. Group Ⅰ (n=10): Separating cell preparations were not purified, Group Ⅱ(n=10): Islet sediment was purified with 25% Ficoll400 ,Group Ⅲ (n=10): Islet sediment was purified with 25% and 11% Ficoll-400. The levels of protein of CK-19, PDX-1, Nestin and Ngn3 were detected by immunohistochemistry and the mRNA of CK-19, PDX-1, Nestin, Ngn3 was amplified by RT-PCR. Results: After two different purification methods applied, three islet preparations of different purities were obtained. The difference of islet purity was significant among various groups (P＜0.05). Compared with group Ⅱ and group Ⅲ,the protein and mRNA of CK-19, PDX-1, Nestin,Ngn3 were both higher in group Ⅰ; group Ⅲ was poorly expressed. Conclusions: The three different islet purity donor islet have different CK-19, PDX-1, Nestin, Ngn3 positive cells within them, indicating that there are some islet stem cells in the purified donor islet.     

Dr.Zhang Ren''''s Experience in the Acupuncture Treatment of Different Diseases with the Same Therapeutic Principle

Dr.Zhang Ren,the chief physician,is the chairman of Shanghai Acupuncture and Moxibustion Association.Having been engaged in medicine for about 40 years,he is experienced in treating various intractable diseases.In his long years of clinical practice,he advocates taking the TCM differentiation as the basis to seek for the acupuncture method for treatment of modern intractable diseases.The author of this essay had the fortune to follow Dr.Zhang in study.The following is a summary of Dr.Zhang's experience in the acupuncture treatment for different intractable diseases with the same therapeutic principle.     

Luo Lingjie''''s Experience in Treating Chronic Nephropathy

In treating chronic nephropathy,Luo Lingjie,a chief physician,pays attention to regulating the balance between yin and yang,treating infection if present,and removing pathogenic factors.He prescribes gentle drugs and uses carefully strongly warming-tonifying ones,emphasizes the importance of persuading the patient to persist in treatment with medication and nurse one's health for recuperation,and is good at combined use of TCM and western medicine therapy and brings the merits of various therapies into full play,with obvious theraoeutic effects.     

Acupuncture Combined with Spinal Tui Na for Treatment of Primary Dysmenorrhea in 30 Cases

Objective: To observe the therapeutic effects in acupunture treatment of primary dysmenorrhea combined with spinal Tui Na, and study its mechanism. Methods: Thirty cases of the treatment group were treated by acupuncture combined with spinal Tui Na, and thirty cases in the control group were treated by routine acupuncture. Results: The total effective rate was 93.3% in the treatment group, and 73.3% in the control group, with a significant difference between the two groups (P<0.05). Conclusions: Acupuncture combined with spinal Tui Na has good prospects for treatment of primary dysmenorrhea.