三七总皂甙对神经干细胞诱导分化多巴胺能神经元移植帕金森病大鼠的影响研究

目的 探讨三七总皂甙对神经干细胞体外诱导分化多巴胺能神经元移植帕金森病(PD)大鼠后移植细胞的存活及移植疗效的影响.方法 大鼠胚胎中脑神经干细胞经传代扩增后,在分化液中诱导向多巴胺能神经元分化,应用6-羟基多巴胺制备的PD大鼠模型随机分为多巴胺能神经元组、多巴胺能神经元 三七总皂甙组、三七总皂甙组及手术对照组,每组8只,进行移植手术,移植后检测PD大鼠不对称旋转行为的变化及纹状体移植区酪氨酸羟化酶染色阳性细胞存活的情况.结果 与手术对照组比较,移植后20 d多巴胺能神经元组大鼠不对称旋转圈数开始明显下降(P＜0.01).移植后10～60 d,多巴胺能神经元 三七总皂甙组大鼠的不对称旋转圈数明显低于多巴胺能神经元组(P＜0.01).免疫组织化学染色显示多巴胺能神经元 三七总皂甙组大鼠纹状体移植区酪氨酸羟化酶染色阳性细胞数明显多于多巴胺能神经元组(P＜0.01).结论 三七总皂甙具有提高神经干细胞诱导分化的多巴胺能神经元移植PD大鼠后移植细胞的存活率及移植疗效的作用.     

基因治疗帕金森病大鼠脑内纹状体多巴胺含量的检测

目的观察脑源性神经营养因子（ＢＤＮＦ）基因工程成肌细胞脑内纹状体移植对帕金森病（ＰＤ）大鼠脑内纹状体多巴胺含量的影响。方法建立逆转录病毒介导的ＢＤＮＦ表达质粒,并转染成肌细胞进行ＰＤ大鼠脑内纹状体移植。结果细胞移植后第２和第８周,移植组毁损侧纹状体多巴胺含量〔分别为（９５７５３±８８９５）和（１０４０２９±１０４７８）ｐｇ／ｍｇ〕较对照组〔分别为（３３５９８±１０２４８）和（３２７８８±７０２３）ｐｇ／ｍｇ〕明显增加（均为Ｐ＜００１）,并可维持２个月之久。结论脑源性神经营养因子基因工程成肌细胞脑内纹状体移植,可使脑内纹状体多巴胺含量明显增加,可能为帕金森病的治疗提供了一种新的治疗方法。     

帕金森病治疗进展

帕金森病的治疗近几年进展迅速，无论在神经保护剂，神经营养剂，多巴胺能类药物，脑深部刺激等方面的研究都取得了长足的进步，免疫机制以及人脑或胎脑移植治疗帕金森病尚处于研究阶段，本文通过对帕金森病的治疗进展作一综述，旨在指导帕金森病病人的合理治疗。     

脑出血大鼠脑内胚胎神经干细胞移植的运动神经功能改变和形态学研究

目的研究脑出血大鼠脑内胚胎神经干细胞移植对运动神经功能改善作用以及移植细胞分化后形态学改变。方法通过大鼠尾状核注射胶原酶IV制作脑出血模型大鼠,分离培养胚胎神经干细胞移植入脑出血大鼠脑内。对脑出血大鼠移植前后运动神经功能进行评价,并通过组织免疫荧光检测移植神经干细胞脑内分化后形态学改变情况。结果Hoechst标记的神经干细胞移植入脑出血大鼠后可见其在脑内存活,主要分布于血肿腔周边,免疫荧光检测显示移植细胞能进一步分化为神经元及星形胶质细胞;从移植术后第21天到第28天,神经干细胞移植组大鼠的神经功能改善显著好于培养液移植组和单纯脑出血组,有统计学意义(P<0.001)。结论胚胎神经干细胞脑出血大鼠脑内移植能促进偏瘫肢体功能恢复;胚胎神经干细胞脑出血大鼠脑内移植后能存活并进一步分化为神经元及星形胶质细胞。     

应用微透析技术活体检测帕金森病动物模型的神经生化改变

目的以活体检测探讨帕金森病（ＰＤ）猴模型神经递质含量的改变。方法应用１－甲基－４－苯基－１，２，３，６－四氢吡啶（ＭＰＴＰ）制备偏侧ＰＤ猴模型，采用颅内微透析技术结合高效液相色谱－电化学方法（ＨＰＬＣ－ＥＣ）活体检测双侧尾核区多巴胺（ＤＡ）及其代谢产物的含量。结果ＭＰＴＰ注射侧与注射对侧相比，尾核区ＤＡ、３，４－二羟基苯乙酸（ＤＯＰＡＣ）和高香草酸（ＨＶＡ）的含量分别减少７０％、３４％和３８％。结论颅内微透析技术是活体检测脑内神经递质含量的最有效方法，可以直接反映出与ＰＤ样症状有关的细胞外液神经递质变化     

成年犬自体骨髓基质干细胞经冠状动脉移植在心梗后心肌组织内分化

目的　探讨经冠状动脉移植成年犬自体骨髓基质干细胞在心梗后心肌组织内的存活、分化情况。方法　结扎犬冠状动脉前降支制备心肌梗死模型 ;细胞移植组将体外培养、诱导的自体骨髓基质干细胞标记后在心肌梗死后 4周经冠状动脉植入心脏 ;对照组植入等量培养基 ;细胞移植 4周后取犬心脏 ,行组织学检查、免疫组织化学检查及电镜检查。结果　细胞移植组植入骨髓基质干细胞在心肌组织内存活 ;免疫组织化学检查结蛋白、肌钙蛋白I染色阳性 ;电镜示心肌疤痕组织中可见胞浆丰富的异源性细胞 ,圆形或椭圆形 ,细胞体积较大 ,核糖体丰富 ,线粒体较丰富 ,有肌丝样结构 ,单核。对照组心脏标本中可见疤痕组织 ;心肌细胞结蛋白染色阴性 ,肌钙蛋白I染色阳性 ;电镜未发现异源性细胞。结论　成年犬骨髓基质干细胞经冠状动脉移植后在心肌组织内存活并分化为肌源性细胞。     

免疫耐受模型的建立与胰岛移植的研究

本研究试图建立供体特异性的免疫耐受动物模型，并探索胰岛移植与免疫耐受的关系，探讨移植区生理与胰岛存活和功能的关系。结果发现：（１）从免疫应答和皮肤，胰移植物存活 经胸腺或门脉植入细胞抗原，是建立免疫耐受的有效途径；植入细胞抗原和联合应用免疫抑制剂可诱导免疫耐受；（２）在建立免疫耐受模型基础上，发现脾内或肝胰岛移植物有功能存活期明显延长，且渡过了急性排斥期；     

帕金森病食蟹猴模型回肠结构与功能相关蛋白变化的研究

目的 探究帕金森病食蟹猴模型研究其肠道结构与功能的变化,为帕金森病患者出现便秘等肠道症状的机制及其干预提供科学依据。方法 选取6只雄性食蟹猴,其中3只使用颈内动脉注射1-甲基-4-苯基-1,2,3,6-四氢吡啶的方式进行造模并出现帕金森病运动症状的食蟹猴,根据Kurlan评分>10分且持续3个月未出现消退迹象为帕金森病组,另外3只未经干预的食蟹猴为对照组。收集2组脑组织与回肠组织,用免疫组织化学染色法酪氨酸羟化酶(TH)分析脑内黑质区多巴胺能神经元数量以及肠道外周TH变化,用苏木精-伊红染色分析肠道的结构,并用闭锁小带蛋白1(ZO-1)评估肠道屏障功能,肠道起搏细胞标志物原癌基因(c-kit)、蛋白基因产物9.5(PGP 9.5)评估肠道蠕动功能。结果 帕金森病组左、右两侧黑质多巴胺能神经元数目明显低于对照组[(133.13±108.63)个vs(957.21±225.56)个,t=5.705,P=0.005;(155.74±62.48)个vs(917.52±161.14)个,t=7.611,P=0.002],对照组回肠肌间神经丛TH吸光度值明显高于帕金森病组(0.79±0.01...     

人胚胎脑细胞在大鼠脑梗死灶内的分化和整合

目的 探讨人胚胎脑细胞在脑梗死灶内的分化、整合状况和神经细胞移植治疗脑梗死的可能性。方法 将正常人胚胎脑细胞和胶质细胞混合培养后，移植到免疫抑制的11只肾性高血压大鼠的大脑皮质梗死灶内，并与未移植的5只脑梗死大鼠作对照。8周后取大鼠脑组织做免疫组化检查。结果移植了人胚胎脑细胞存活的lO只大鼠中，6只有移植物生长。移植物内有新生血管，细胞有分层排列趋势。免疫组化染色证实，移植物内有细胞分化，并存在大量神经元和星形胶质细胞。神经元的树突和轴突汇集成束，整合到宿主脑内。结论 培养的人胚胎脑细胞能在免疫抑制大鼠的脑梗死灶内生长、分化和整合，提示神经细胞移植有望成为临床治疗脑梗死的突破性方法。     

脑内胰岛移植治疗Ⅰ型糖尿病的远期疗效观察

应用短期培养的人胎儿胰岛组织脑内移植治疗Ⅰ型糖尿病患者３０例，年内有效２６例（８７％）；移植后２～３年随访２１例，有效１７例（８１％），失效４例（１９％）；４～５年随访１８例，有效１５例（８３．３％），失效３例（１６．７％）；６～７年随访１５例，有效９例（６０％），失效６例（４０％）。在移植有效病例中，有效维持时间为４．９±１．７（１．２～７）年，１１例完全或间断停用胰岛素治疗达０．３～７（３．１２±１．９６）年，其中超过３年者６例。结果表明，脑内胰岛移植治疗Ⅰ型糖尿病临床效果显著，移植物在部分患者脑内能长期存活。     

大鼠脑出血后神经干细胞移植的实验研究

目的探讨脑出血后大鼠胚胎神经干细胞移植治疗的可行性.方法从14d胚龄的大鼠胚胎脑组织中分离、培养神经干细胞,通过荧光免疫组化技术研究其特性.制作大鼠脑出血模型,分别于3d和7 d时将未分化的神经干细胞注入血同侧的尾状核内.分别记录模型制作后2 h和移植后2 h、4周的大鼠运动功能.移植4周后处死大鼠,观察移植后干细胞在体内生长情况.结果实验中分离、培养的神经干细胞体外能够被诱导分化成神经元、少突胶质细胞和星形胶质细胞.出血后7 d干细胞移植组的大鼠运动功能的改善显著好于3 d组和对照组.结论神经干细胞移植治疗能够显著改善脑出血动物的运动功能,是一种很有发展前途的方法,值得进行更深入的研究.     

Neuronal cell replacement in Parkinson’s disease

Transplantation of foetal dopamine neurons into the striatum of Parkinson’s disease patients can provide restoration of the dopamine system and alleviate motor deficits. However, cellular replacement is associated with several problems. As with pharmacological treatments, cell therapy can lead to disabling abnormal involuntary movements (dyskinesias). The exclusion of serotonin and GABA neurons, and enrichment of substantia nigra (A9) dopamine neurons, may circumvent this problem. Furthermore, although grafted foetal dopamine neurons can survive in Parkinson’s patients for more than a decade, the occurrence of Lewy bodies within such transplanted cells and reduced dopamine transporter and tyrosine hydroxylase expression levels indicate that grafted cells are associated with pathology. It will be important to understand if such abnormalities are host‐ or graft induced and to develop methods to ensure survival of functional dopamine neurons. Careful preparation of cellular suspensions to minimize graft‐induced inflammatory responses might influence the longevity of transplanted cells. Finally, a number of practical and ethical issues are associated with the use of foetal tissue sources. Thus, future cell therapy is aiming towards the use of embryonic stem cell or induced pluripotent stem cell derived dopamine neurons.     

Visualization, direct isolation, and transplantation of midbrain dopaminergic neurons

To visualize and isolate live dopamine (DA)-producing neurons in the embryonic ventral mesencephalon, we generated transgenic mice expressing green fluorescent protein (GFP) under the control of the rat tyrosine hydroxylase gene promoter. In the transgenic mice, GFP expression was observed in the developing DA neurons containing tyrosine hydroxylase. The outgrowth and cue-dependent guidance of GFP-labeled axons was monitored in vitro with brain culture systems. To isolate DA neurons expressing GFP from brain tissue, cells with GFP fluorescence were sorted by fluorescence-activated cell sorting. More than 60% of the sorted GFP(+) cells were positive for tyrosine hydroxylase, confirming that the population had been successfully enriched with DA neurons. The sorted GFP(+) cells were transplanted into a rat model of Parkinson's disease. Some of these cells survived and innervated the host striatum, resulting in a recovery from Parkinsonian behavioral defects. This strategy for isolating an enriched population of DA neurons should be useful for cellular and molecular studies of these neurons and for clinical applications in the treatment of Parkinson's disease.     

Dopamine action and disorders of neurotransmitter balance

Disorders of neurotransmitter balance are observed in Parkinson's disease, pharmacotoxic psychosis and depression. The dopamine-serotonin ratio is reduced to about 20% in Parkinson and pharmacotoxic patients in the caudate nucleus and in the substantia nigra. The serotonin content in these brain areas is lowered only to about 50% in comparison to that of the control, whereas the dopamine level is reduced to 85% in Parkinson patients. This dopamine deficiency has been substituted by exogenous supply of L-dopa in combination with decarboxylase and monoaminooxydase inhibitors. First evidence is presented that L-dopa can be replaced, at least partially, by iron in form of a ferriascorbate complex. This iron compound improves the symptoms of Parkinson's disease to almost the same extent as L-dopa.     

骨髓间充质干细胞在脑缺血再灌注大鼠脑内存活并分化为神经元样细胞的实验研究

目的观察骨髓间充质干细胞(MSCs)经静脉移植在大脑中动脉缺血再灌注(MCAO)大鼠脑内存活并分化为神经元样细胞。方法常规方法分离、培养大鼠MSCs,根据Aspey方法制成MCAO模型,经尾静脉注射3×106溴脱氧尿嘧啶(BrdU)标记的大鼠MSCs,28d后处死大鼠,取脑组织行免疫荧光检测。结果共聚焦显微镜下观察到MSCs移植后脑梗死灶边缘聚大量BrdU阳性细胞,少量神经元特异性烯醇化酶(NSE)和BrdU双染细胞。结论经静脉移植的MSCs可移行至MCAO脑梗死灶周围并分化为神经元样细胞。     

Personality traits and brain dopaminergic function in Parkinson's disease.

A distinctive personality type, characterized by introversion, inflexibility, and low novelty seeking, has been suggested to be associated with Parkinson's disease. To test the hypothesis that Parkinson's disease is associated with a specific dopamine-related personality type, the personality structures of 61 unmedicated Parkinson's disease patients and 45 healthy controls were examined. Additionally, in 47 Parkinson's disease patients, the dopaminergic function in the brain was directly measured with 6-[(18)F]fluoro-l-dopa ((18)F-dopa) positron emission tomography (PET) with MRI coregistration. The novelty-seeking personality score, supposedly associated with the parkinsonian personality, was slightly lower in the Parkinson's disease group compared with controls, but it did not have a significant relationship with (18)F-dopa uptake in any of the brain regions studied (r = -0.12 to 0.11, P > 0.15). The harm-avoidance personality score, associated with anxiety and depression, was clearly increased in patients with Parkinson's disease and it had a paradoxical, highly significant positive correlation with the (18)F-dopa uptake in the right caudate nucleus (r = 0.53, P = 0.04, Bonferroni corrected for 220 comparisons). Although the results of this study are not in disagreement with the concept of low-novelty-seeking personality type in Parkinson's disease, the personality type does not seem to be dopamine dependent. The correlation between the personality trait of harm avoidance and (18)F-dopa may reflect a specific feedback circuitry of neurotransmitters that is associated with negative emotionality in Parkinson's disease.     

Interrelationship between Activation of Dopaminergic Pathways and Cerebrospinal Fluid Concentration of Dopamine Tetrahydroisoquinoline Metabolite Salsolinol in Humans: Preliminary Findings

The main objective of this study was to determine whether the activation of dopaminergic pathways, through adrenal-caudate transplantation, stimulated the production of dopamine and salsolinol in cerebrospinal fluid (CSF) of patients with Parkinson's disease. Dopamine sulfate and salsolinol sulfate in CSF specimens were measured by radioenzymatic technique. The results of this study demonstrated that the replacement of degenerative nigrostriatal neurons with new dopamine-producing cells by adrenal brain transplants in patients with Parkinson's disease resulted in significant increase (p less than 0.05) in CSF levels of free dopamine, dopamine sulfate, free salsolinol, and salsolinol sulfate as compared with preoperative levels. Moreover, the oral administration of L-dopa to these transplanted patients caused substantial (p less than 0.001) elevation in CSF levels of free dopamine (before L-dopa, 146 +/- 57 pg/ml; after L-dopa, 575 +/- 207 pg/ml), dopamine sulfate (before L-dopa, 1966 +/- 945 pg/ml; after L-dopa, 41679 +/- 29326 pg/ml), free salsolinol (before L-dopa, 43 +/- 29 pg/ml; after L-dopa, 186 +/- 90 pg/ml), and salsolinol sulfate (before L-dopa, 405 +/- 477 pg/ml; after L-dopa, 2908 +/- 2572 pg/ml), respectively.     

Liver transplantation for hepatitis C virus related cirrhosis.

Hepatitis C virus (HCV) related chronic liver disease is now the leading cause for liver transplantation in many centres. Virological recurrence is inevitable following liver transplantation. Excellent patient and graft survival are seen in the short-term, equivalent to that in patients transplanted for other causes of liver disease. However, histological evidence of disease recurrence or hepatitis is present in over half the patients within a year of transplantation, although a small percentage develop progressive cholestatic hepatitis with graft loss within a year. Cirrhosis can develop in the first year after transplantation and 28% of patients have evidence of cirrhosis by 5 years. There is little agreement over the factors that predict the recurrence of disease, development of cirrhosis within the graft and graft or patient survival. Graft loss due to HCV occurs in up to 9% at 5 years and the long-term prognosis may not be comparable to groups transplanted for other diseases. Patients with hepatocellular carcinoma may benefit from liver transplantation if the tumour is small and without vascular invasion. There are, as yet, no clear guidelines regarding the best combination of immunosuppressants in patients with HCV but viral clearance has been achieved with the use of interferon and ribavirin therapy post-operatively.