微囊化PC12细胞移植后帕金森病大鼠旋转行为变化研究

目的观察微囊化PC12细胞移植后帕金森病大鼠旋转行为的变化。方法采用免疫隔离微囊技术将能分泌多巴胺的PC12细胞包裹后,移植到帕金森病大鼠脑内,观察阿扑吗啡诱发病鼠旋转行为的变化,酪氨酸羟化酶(TH)抗体免疫组化染色观察移植区阳性细胞的表达。结果帕金森病大鼠旋转行为有明显改善,由移植前(11.88±2.04)圈/min减少为移植后(3.46±1.01)圈/min。移植后3个月,大鼠脑内微囊完整,仍有TH阳性PC12细胞存活。结论微囊化PC12细胞移植可明显改善帕金森病大鼠的旋转行为。     

微囊化PC12细胞移植治疗帕金森病的实验研究

目的　研究治疗帕金森病的一种新方法。方法　采用免疫隔离技术－微囊技术将能分泌多巴胺的ＰＣ１２细胞包裹后,移植到帕金森病大鼠脑内,观察阿扑吗啡诱发病鼠旋转行为变化,高效液相色谱电化学法测定移植区多巴胺含量,酪氨酸羟化酶（ＴＨ）抗体免疫组织化学染色观察移植区阳性细胞表达。结果　帕金森病大鼠阿扑吗啡诱发旋转行为明显改善,不同时相点的移植区多巴胺含量较对照组明显升高,而未行微囊化的ＰＣ１２细胞移植则会有致死性肿瘤形成。移植后３月,大鼠脑内微囊完整,仍有ＴＨ阳性ＰＣ１２细胞存活。结论　微囊化ＰＣ１２细胞移植,可避免药物治疗、立体定向毁损手术、胎脑移植等方法的诸多弊端,因此是一种新的有效的治疗帕金森病的方法。     

微囊化PC12细胞脑内移植治疗帕金森病的实验研究

目的观察微囊化PC12细胞脑内移植对帕金森病(PD)大鼠模型的治疗作用。方法以6-OHDA分两点注入大鼠纹状体制备PD模型。应用国产新型材料壳聚糖制成的海藻酸钠一壳聚糖一海藻酸钠(ACA)微囊包裹PC12细胞，分别将微囊化PC12细胞、裸PC12细胞、空微囊移植入PD模型鼠损伤侧纹状体内，以阿朴吗啡检测移植前后大鼠旋转行为的差异。行纹状体和黑质的HE染色和酪氨酸羟化酶免疫组化染色观察其病理形态学的变化。对移植的微囊化PC12细胞回收后再培养，以MTT和台盼兰检测细胞活性。结果微囊化PC12细胞移植能够改善PD模型鼠的旋转行为，与移植前和空微囊移植组相比有显著差异(P＞O．05)，症状改善至少持续3个月。裸PC12细胞移植组大鼠的旋转行为也有改善，与移植前相比有统计学差异(P＜O．05)，但仅持续了2个月，移植8周后又渐回到移植前水平，且部分大鼠颅内有致死性肿瘤形成。空微囊移植组移植前后大鼠的旋转行为无明显差异(P＞O．05)。回收微胶囊内的PC12细胞再培养生长良好，MTT法和台盼兰法检测显示细胞具有生物活性。结论微囊化PC12细胞脑内移植能够改善阿朴吗啡诱发的PD模型鼠的旋转症状，ACA新型微包囊能够起到有效的免疫隔离和抑制肿瘤形成的作用。     

胚胎多巴胺神经元移植治疗帕金森病的实验研究

目的探讨胚胎多巴胺神经元移植对帕金森病大鼠的治疗作用。方法立体定向注射6-羟多巴胺建立帕金森病大鼠模型,随机分为对照组(n=12)和细胞移植组(n=12)。细胞移植组将荧光染料CM-DiI标记的大鼠胚胎多巴胺神经元立体定向注入帕金森病大鼠纹状体区,对照组于相同部位注入生理盐水。用阿扑吗啡诱导帕金森病大鼠旋转行为评估细胞移植的治疗作用。细胞移植8周后取大脑标本行冷冻切片,荧光显微镜下观察移植细胞在脑内存活情况。结果与对照组比较,移植多巴胺神经元能显著改善阿扑吗啡诱导帕金森病大鼠的异常旋转行为(P0.01)。移植8周后,仅少量多巴胺神经元存活。结论多巴胺神经元移植可短期内改善帕金森病大鼠的运动障碍,但长期疗效不佳,可能与移植多巴胺神经元长期存活率较低有关。     

牛嗜铬细胞脑内移植可减少帕金森病样大鼠药物诱发的旋转行为

目的观察牛嗜铬细胞移植于PD样大鼠脑内的存活及功能。方法用6－OHDA损毁大鼠一侧黑质细胞制成偏侧PD样大鼠模型。在右侧纹状体内植入约2万个牛嗜铬细胞悬液。记录阿朴吗啡诱发大鼠的旋转行为。实验结束后取大鼠脑做组织形态学检查。结果大鼠在移植后阿朴吗啡诱发的旋转行为得到明显纠正（P<0．001）。移植后12周荧光组化显示移植区有大量SPG阳性细胞，其形态类似在正常肾上腺髓质内的嗜铬细胞的形态。结论脑内移植牛嗜铬细胞可改善PD样大鼠药物诱发的旋转行为；提示牛嗜铬细胞在移植12周后仍能在大鼠脑纹状体内存活。     

微囊化牛视网膜色素上皮细胞移植治疗伴Lewy小体形成帕金森病的实验研究

目的观察微囊化牛视网膜色素上皮细胞(RPE)移植对伴Lewy小体形成帕金森病(PD)大鼠的治疗作用。方法用蛋白酶体选择性抑制剂lactacystin制作伴Lewy小体形成的PD模型;将微囊化牛RPE植入大鼠纹状体,移植分生理盐水对照组(NS)、RPE、空微囊(APA)及微囊化RPE(RPE-APA)四组;观察移植后旋转行为变化、纹状体中多巴胺(DA)和高香草酸(HVA)及酪氨酸羟化酶(TH)含量的变化。结果微囊化RPE的旋转行为在移植后第1周开始改善(改善程度为21.3%,与空微囊组相比P<0.05),第4周改善更加明显(改善程度为57.89%,与第1周相比P<0.05),并一直持续到第14周(改善程度为59.47%,与空微囊组相比P<0.05)。行为学改善的大鼠,纹状体内DA和HVA含量的变化同其行为学改善相符合。行为学有改善大鼠囊内细胞TH染色呈弱阳性,微囊周边的纹状体可见TH阳性纤维密度较空微囊组高。结论微囊化牛RPE脑内移植对伴Lewy小体形成PD大鼠有治疗作用。     

微囊化人视网膜色素上皮细胞移植治疗帕金森病的实验研究

目的 观察微囊化人视网膜色素上皮(RPE)细胞移植治疗帕金森病(PD)大鼠的疗效. 方法 采用机械分离法和酶消化法原代培养人RPE细胞,传代后用高压静电微胶囊成型装置制作海藻酸钠-多聚赖氨酸-海藻酸钠微囊化细胞,将其立体定向移植人6-羟基多巴胺(6-OHDA)所致的PD模型大鼠的右侧纹状体.实验分为模型组、裸细胞(RPE)组、空囊对照(APA)组以及微囊化细胞(APA-RPE)组.检测各组大鼠移植前后阿朴吗啡诱导的旋转行为变化和移植后8周纹状体中多巴胺(DA)的含量. 结果 APA-RPE组大鼠在移植后4周阿朴吗啡诱发的旋转次数[(6.25±1.04)r/min]开始减少,与移植前[(12.88±7.34)r/min]相比减少幅度为51.48%,至第8周[(5.87±2.03)r/min]减少更加明显,减少幅度为54.43%,差异均有统计学意义(P<0.05);与模型组[(108.14±1.89)mol/L]比较,APA-RPE组移植后8周[(342.63±28.32)mol/L]大鼠纹状体DA含量明显增加,差异有统计学意义(P<0.05),而RPE组和APA组未见明显变化. 结论 微囊化人RPE细胞对PD大鼠模型有治疗作用,可作为一种前景良好的治疗PD的方法 进一步研究.     

GFP转基因小鼠神经干细胞移植治疗大鼠帕金森病的实验研究

目的观察GFP(绿色荧光蛋白)转基因小鼠胚胎神经干细胞植入帕金森病大鼠纹状体后的存活、分化情况及治疗作用。方法建立PD模型大鼠及体外培养神经干细胞,然后将GFP转基因小鼠神经干细胞定向植入帕金森病大鼠毁损侧纹状体内,于移植后不同时间诱发旋转行为,并与对照组相比,观察症状的改善,并用酪氨酸羟化酶(TH)免疫组织化学染色方法检测移植GFP转基因小鼠神经干细胞的存活及分化状况。结果 GFP转基因小鼠神经干细胞脑内移植后,帕金森病大鼠的旋转行为明显改善。移植后2至4周时可检测到成片或散在的TH免疫阳性细胞。结论 GFP转基因小鼠神经干细胞移植至帕金森病大鼠纹状体后,可分化为多巴胺能神经元并能改善旋转症状。     

帕金病样大鼠模型的稳定性观察

6-OHDA损毁大鼠一侧中脑黑质细胞造成偏侧帕金森病(PD)样大鼠模型,用小动物旋转行为记录仪记录阿朴吗啡诱发大鼠的旋转行为;TH免疫细胞化学染色观察中脑黑质细胞的损毁状况。连续观察10个月其旋转行为无自发性恢复;PD样模型大鼠损毁侧中脑无TH阳性细胞存在。结果表明6-OHDA损毁大鼠一侧中脑黑质细胞可造成稳定、可靠的类似PD病人病理变化的偏侧动物模型。     

铁离子对黑质纹状体多巴胺神经元毒性作用的实验研究

目的 探讨铁离子对黑质纹状体多巴胺神经元的毒性作用。方法 采用立体定向偏侧大鼠黑质内注入50μg FeCl3和FeCl2，4周后用阿朴吗啡诱导动物行为学变化，高效液相色谱(HPLC)检测纹状体内多巴胺、去甲肾上腺素、肾上腺素递质含量的变化，免疫组化观察黑质多巴胺神经元和胶质细胞的改变。结果 FeCL3和FeCL2均可引起注射侧纹状体内DA含量选择性降低，而NA、A含量无显著改变；注射侧黑质内DA神经元显著缺失、胶质细胞显著增生；FeCL3组阿朴吗啡诱导大鼠向同侧旋转行为，FeCL2组大鼠于术后即出现特征性自发性对侧旋转行为，阿朴吗啡不能诱发其旋转。结论 铁离子对黑质纹状体多巴胺神经元具有毒性作用，Fe^3 作用最强，胶质细胞的增生可能参与了这一毒性作用过程。     

Human amniotic epithelial cells produce dopamine and survive after implantation into the striatum of a rat model of Parkinson's disease: a potential source of donor for transplantation therapy

We have recently found that human amniotic epithelial (HAE) cells synthesize catecholamines including dopamine (DA). The present study was designed to explore the possibility of HAE cells to serve as a donor for transplantation therapy of Parkinson's disease (PD). Thus, we investigated their ability to produce DA in vitro and the survival and function of HAE cells grafted into a rat model of PD. RT-PCR and Western blotting revealed that HAE cells express tyrosine hydroxylase (TH) mRNA and protein, respectively. TH-immunohistochemistry on cultured HAE cells demonstrated that around 10% of the total cells are immunopositive for this protein. The production of DA by HAE cells was increased with time in the presence of L-tyrosine and BH(4), and was abolished with a specific TH inhibitor, alpha-methyl-rho-tyrosine. Dissociated HAE cells transduced with the Escherichia coli LacZ marker gene (beta-gal) were implanted into the previously DA-depleted striatum of immunosuppressed rats. Two weeks postgrafting HAE grafts were demonstrated to survive without overgrowth, as evidenced by the presence of beta-gal-positive cells and TH-immunoreactive cells within the grafts. The grafts also provided partial amelioration of apomorphine-induced rotational asymmetry. The results clearly indicate that HAE cells capable of producing DA can survive and function in the brain of a rat model of PD. Although DA replacement therapy of PD could possibly be achieved with implantation of HAE cells, further studies are needed to develop strategies to enhance the ability of HAE cells to produce DA as well as the graft survival.     

基因修饰骨髓源性神经元样干细胞治疗帕金森大鼠的研究

目的 观察大鼠酪氨酸羟化酶(tyrosinehydroxylase,TH)修饰的骨髓基质源性神经元样干细胞(neuronoid stem cells derived from bone marrow stem cells,NdSCs-D-BMSCs)在脑室移植途径下对帕金森病(Parkinson disease,PD)大鼠的治疗作用.方法 将酶切鉴定后的新构建质粒pEGFP-C2-TH经电穿孔法转染培养第8天NdSCs-D-BMSCs,注射到PD大鼠模型右侧脑室,观察大鼠行为学变化,移植细胞在大鼠脑组织内的迁移,以及高效液相方法检测脑内DA含量.结果 质粒pEGFP-C2-TH转染NdSCs-D-BMSCs移植后10周,PD大鼠症状显著改善,DA恢复至正常水平33.0%,移植细胞可以在PD大鼠脑内存活,并出现远处迁移.结论 TH修饰的大鼠NdSCs-D-BMSCs经脑室移植对PD大鼠具有明显的治疗作用,为临床中腰椎穿刺干细胞移植的应用提供实验依据.     

Relationships between various behavioural abnormalities and nigrostriatal dopamine depletion in the unilateral 6-OHDA-lesioned rat

While rotational asymmetry is used as a characteristic behavioural sign of striatal dopamine (DA) loss in unilateral animal models of Parkinson's disease (PD), there is relatively little analysis of how other common behavioural deficits relate to nigrostriatal DA depletion. We analysed the relationships between several deficits induced by unilateral 6-OHDA lesions and striatal neurochemistry, as well as neuronal loss in the dopaminergic substantia nigra (SN). Behaviour was evaluated from before until 6 weeks after surgery and abnormalities appeared in body axis, head position and sensorimotor performance as well as apomorphine-induced rotation. As expected, rotational behaviour correlated with striatal DA loss and not with other striatal neurotransmitters measured. Similar observations were found for sensorimotor deficits ('disengage task'). Both deficits were observed in rats with >70% loss of TH+ nigral neurons and >80% loss of striatal DA. Additional postural abnormalities appeared with mean losses of 87% of nigral DA neurons and 97% striatal DA, consistent with observations in patients with advanced PD. The data show that the repertoire of behavioural abnormalities manifested by hemiparkinsonian rats relate directly to the degree of nigrostriatal DA loss and, therefore, mimic features of PD. Analysis of such behaviours are relevant for chronic therapeutic studies targeting PD.     

Intranigral transplantation of solid tissue ventral mesencephalon or striatal grafts induces behavioral recovery in 6-OHDA-lesioned rats

Parkinson's disease (PD) is characterized by a degeneration of the dopamine (DA) pathway from the substantia nigra (SN) to the basal forebrain. Prior studies in unilateral 6-hydroxydopamine (6-OHDA)-lesioned rats have primarily concentrated on the implantation of fetal ventral mesencephalon (VM) into the striatum in attempts to restore DA function in the target. We implanted solid blocks of fetal VM or fetal striatal tissue into the SN to investigate whether intra-nigral grafts would restore motor function in unilaterally 6-OHDA-lesioned rats. Intra-nigral fetal striatal and VM grafts elicited a significant and long-lasting reduction in apomorphine-induced rotational behavior. Lesioned animals with ectopic grafts or sham surgery as well as animals that received intra-nigral grafts of fetal cerebellar cortex showed no recovery of motor symmetry. Subsequent immunohistochemical studies demonstrated that VM grafts, but not cerebellar grafted tissue expressed tyrosine hydroxylase (TH)-positive cell bodies and were associated with the innervation by TH-positive fibers into the lesioned SN as well as adjacent brain areas. Striatal grafts were also associated with the expression of TH-positive cell bodies and fibers extending into the lesioned SN and an induction of TH-immunolabeling in endogenous SN cell bodies. This finding suggests that trophic influences of transplanted fetal striatal tissue can stimulate the re-expression of dopaminergic phenotype in SN neurons following a 6-OHDA lesion. Our data support the hypothesis that a dopaminergic re-innervation of the SN and surrounding tissue by a single solid tissue graft is sufficient to improve motor asymmetry in unilateral 6-OHDA-lesioned rats.     

A potential target for the treatment of Parkinson's disease: Effect of lateral habenula lesions

ObjectiveParkinson's disease (PD) is a progressive neurodegenerative movement disorder that is caused predominantly by the degeneration of the nigrostriatal dopaminergic pathway. Lateral habenula (LHb) has efferent projections that terminate in the substantia nigra pars compacta (SNpc) and electrical stimulation of the LHb effectively suppresses the activity of dopamine-containing neurons in the SNpc. This study was aimed to investigate whether LHb lesions can ameliorate the syndromes of PD via affecting the activities of SNpc neurons in 6-hydroxydopamine (6-OHDA)-induced PD model rats.MethodsConcentrations of dopamine (DA) and its metabolites dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the striatum, which is the area projected by the SNpc dopaminergic neurons were assayed by high-performance liquid chromatography (HPLC) coupled with fluorescence detection. The immunohistochemical method was applied to detect the numbers of tyrosine hydroxylase (TH)-positive cells in the substantia nigra.ResultsThe results showed that LHb lesions induced a significant reduction in apomorphine-induced rotational behavior. The DA, DOPAC and HVA levels in the striatum of PD model rats were increased by the LHb lesions.ConclusionTherefore, we speculate that the LHb lesions induced a significant amelioration in motor disorders via increasing the DA levels in the striatum, which may lead to a potential therapeutic strategy for the treatment of PD.     

Transient improvement of amphetamine-induced rotational behavior by PC12 cell grafts: studies with microdialysis

The effects of striatal transplantation of PC12 cells on amphetamine-induced rotational behavior and monoamine levels were examined in rats with unilateral 6-hydroxydopamine (6-OHDA) lesions in the nigrostriatal pathway. A total of 9 × 104 or 9 × 105 cells of PC12 was implanted into 3 sites in the striatum and changes in amphetamine (3 mg/kg, i.p.)-induced rotational behavior were observed for 8 weeks. Two weeks after transplantation, a significant reduction in rotation was observed. However, this improvement disappeared after 3 weeks. Three of 7 rats implanted with 9 × 104 cells and 6 of 7 rats with 9 × 105 cells died between 3 and 4 weeks after transplantation. In 6-OHDA-injected control rats, the levels of dopamine (DA) and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in brain dialysates were profoundly reduced to between 0 and 11% of normal rats, while the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) level remained unchanged. These reductions in DA and its metabolites did not recover at 2 or 8 weeks after the transplantation of 9 × 104 PC12 cells. 5-HIAA was reduced at 2 weeks and recovered to nearly control levels at 8 weeks. Histologically, PC12 cells proliferated to form a large tumor mass at 2 weeks. There were almost no processes growing from the aggregated PC12 cells into the host tissue. These results indicate that PC12 grafts do not release detectable quantities of DA into the deafferented striatum, and suggest that the transient improvements in rotational behavior may be due to a non-specific suppression in neuronal function induced by the growing tumor mass.     

TH基因修饰的神经干细胞移植治疗帕金森病的实验研究

目的 探讨TH基因修饰的神经干细胞脑内移植对帕金森病(PD)的治疗作用。方法 构建pN：ATH逆转录病毒载体质粒，用PA317细胞包装，G418筛选阳性克隆，病毒上清感染神经干细胞，将表达TH的神经干细胞植入：PD大鼠纹状体内，测定：PD大鼠旋转行为改善，DA和DOPAC含量变化，以及TH在纹状体的表达。结果 TH基因修饰的神经干细胞移植8周时能显著降低PD大鼠旋转行为，增加纹状体DA和DOPAC含量，TH在纹状体内的表达增加，疗效好于单纯神经干细胞移植组。结论 TH基因修饰的神经干细胞移植对PD大鼠有明显的治疗作用，可望为PD治疗提供新的途径。     

Partial lesion of the substantia nigra: relation between extent of lesion and rotational behavior.

Recent work, largely carried out in primate models of Parkinson's disease (PD), indicates that residual dopaminergic neurons in the midbrain and their axons to the nucleus accumbens and striatum can be stimulated to sprout collateral axons, reinnervate the striatum, and cause a behavioral recovery. We sought to create a partial lesion model of PD in the rat that would (i) mimic the pattern of cell loss in human patients in early stages of PD, and (ii) permit examination of experimental manipulations that promote sprouting of axons of the surviving dopaminergic cells in the midbrain. Rats with unilateral 6-hydroxydopamine (6-OHDA) lesions of the substantia nigra pars compacta (SNpc) were tested weekly for rotational asymmetry following administration of apomorphine or amphetamine. After completion of behavioral testing, the animals were sacrificed and the brains immunolabeled for tyrosine hydroxylase (TH). Analysis of anatomical and behavioral data revealed a strong correlation between number of remaining TH-immunoreactive cells in the SNpc and the number of rotations induced by apomorphine. There was no significant correlation between number of remaining TH-immunoreactive nigral neurons and number of rotations induced by amphetamine. We also examined the relation between area in the denervated striatum with remaining TH-immunoreactive axons, number of TH-immunoreactive cells in the lesioned SNpc, and rotational behavior. As expected, there was a strong correlation between area innervated by TH-immunoreactive axons and number of remaining TH-immunoreactive neurons in the lesioned SNpc. Total extent of innervation was also correlated with number of apomorphine-induced rotations but not with number of amphetamine-induced rotations.(ABSTRACT TRUNCATED AT 250 WORDS)