Impact of Pitx3 gene knockdown on glial cell line-derived neurotrophic factor transcriptional activity in dopaminergic neurons

Pitx3 is strongly associated with the phenotype, differentiation, and survival of dopaminergic neurons. The relationship between Pitx3 and glial cell line-derived neurotrophic factor（GDNF） in dopaminergic neurons remains poorly understood. The present investigation sought to construct and screen a lentivirus expression plasmid carrying a rat Pitx3 short hairpin（sh）RNA and to assess the impact of Pitx3 gene knockdown on GDNF transcriptional activity in MES23.5 dopaminergic neurons. Three pairs of interference sequences were designed and separately ligated into GV102 expression vectors. These recombinant plasmids were transfected into MES23.5 cells and western blot assays were performed to detect Pitx3 protein expression. Finally, the most effective Pitx3 sh RNA and a dual-luciferase reporter gene plasmid carrying the GDNF promoter region（GDNF-luciferase） were cotransfected into MES23.5 cells. Sequencing showed that the synthesized sequences were identical to the three Pitx3 interference sequences. Inverted fluorescence microscopy revealed that the lentivirus expression plasmids carrying Pitx3-sh RNA had 40-50% transfection efficiency. Western blot assay confirmed that the corresponding Pitx3 of the third knockdown sequence had the lowest expression level. Dual-luciferase reporter gene results showed that the GDNF transcriptional activity in dopaminergic cells cotransfected with both plasmids was decreased compared with those transfected with GDNF-luciferase alone. Together, the results showed that the designed Pitx3-sh RNA interference sequence decreased Pitx3 protein expression, which decreased GDNF transcriptional activity.

Intrastriatal glial cell line-derived neurotrophic factors for protecting dopaminergic neurons in the substantia nigra of mice with Parkinson disease

BACKGROUND： Substantia nigra is deep in position and limited in range, the glial cell line-derived neurotrophic factor （GDNF） injection directly into substantia nigra has relatively greater damages with higher difficulty. GDNF injection into striatum, the target area of dopaminergic neuron, may protect the dopaminergic neurons in the compact part of substantia nigra through retrograde transport. OBJECTIVE： To investigate the protective effect of intrastriatal GDNF on dopaminergic neurons in the substantia nigra of mice with Parkinson disease （PD）, and analyze the action pathway. DESIGN： A controlled observation. SETTING： Neurobiological Laboratory of Xuzhou Medical College. MATERIALS： Twenty-four male Kunming mice of 7 - 8 weeks old were used. GDNF, 1-methy1-4-pheny1-1,2,3,6-tetrahydropyridine （MPTP） were purchased from Sigma Company （USA）; LEICAQWin image processing and analytical system. METHODS： The experiments were carded out in the Neurobiological Laboratory of Xuzhou Medical College from September 2005 to October 2006. The PD models were established in adult KunMing mice by intraperitoneal injection of MPTP. The model mice were were randomly divided into four groups with 6 mice in each group： GDNF 4-day group, phosphate buffer solution （PSB） 4-day group, GDNF 6-day group and PSB 6-day group. Mice in the GDNF 4 and 6-day groups were administrated with 1 μ L GDNF solution （20 μ g/L, dispensed with 0.01 mol/L PBS） injected into right striatum at 4 and 6 days after model establishment. Mice in the PSB 4 and 6-day groups were administrated with 0.01 mol/L PBS of the same volume to the same injection at corresponding time points. ② On the 12^th day after model establishment, the midbrain tissue section of each mice was divided into 3 areas from rostral to caudal sides. The positive neurons of tyroxine hydroxylase （TH） and calcium binding protein （CB） with obvious nucleolus and clear outline were randomly selected for the measurement, and the number of positive neurons in unit area was counted. MAIN OUTCOME MEASURES： Number of positive neurons of TH and CB in midbrain substantia nigra of mice in each group. RESULTS： All the 24 mice were involved in the analysis of results. The numbers of TH^＋ and CB^＋ neurons in the GDNF 4-day group （54.33±6.92, 46.33±5.54） were obviously more than those in the PBS 4-day group （27.67±5.01, 21.50±5.96, P 〈 0.01）. The numbers of TH^＋ and CB^＋ neurons in the GDNF 6-day group （75.67±5.39, 69.67±8.69） were obviously more than those in the PBS 6-day group （27.17±4.50, 21.33 ±5.72, P 〈 0.01） and those in the GDNF 4-day group （P 〈 0.01 ）. CONCLUSION： Intrastriatal GDNF can protect dopaminergic neurons in substantia nigra of PD mice, and it may be related to the increase of CB expression.

Establishment and expression of recombinant human glial cell linederived neurotrophic factor and TNF α receptor in human neural stem cells

Objective:To investigate the interference and expression of human glial cell line-derived neurotrophic factor(hCDNF) and soluble TNF alpha(sTMFRⅠ) receptor genes in neural stem cells and to evaluate the roles of these proteins in the genetic treatment of spinal cord injury.Methods:Full-length of GDNF cDNA(538 bp) and sTMFRⅠcDNA(504 bp) were inserted into the early 1 region of adenovirus genomic DNA respectively and were immediated by the human cytomegalovirus(gene promoter/enhancer). These adenoviruses were propagated in HEK293 cells via homologous recombination for 7-10 days in vivo,then they were used to infect human neural stem ceils.The infection and expression of gene were tested under immunofluorescence.ELISA and Westem-blot after 48 hours.Results:Almost all the cultured cells showed the nestin immunofluorescence positive staining,which was the characteristics of neural stem cell.A great quantity of EGFP and KFP were observed in neural stem cells,which indicated the expression of GDNF and sTMFRⅠ.After transfection of GDNF and sTMFRⅠgenes,many neural stem cells show GFAP and tubulin immunofluorescence positive staining,which meant that most neural stem cells differentiated into neuron at that condition.Conclusions:The infective efficiency of adenovirus is greatly acceptable to neural stem cell,thus adenovirus provide a useful vector for exogenous GDNF and sTMFRⅠgenes expressing in neural stem cells,which is useful for differentiation of neural stem cell.

Differential expression of glial cell line-derived neurotrophic factor splice variants in the mouse brain

Glial cell line-derived neurotrophic factor(GDNF) plays a critical role in neuronal survival and function. GDNF has two major splice variants in the brain,α-pro-GDNF and β-pro-GDNF, and both isoforms have strong neuroprotective effects on dopamine neurons. However, the expression of the GDNF splice variants in dopaminergic neurons in the brain remains unclear. Therefore, in this study, we investigated the mRNA and protein expression of α-and β-pro-GDNF in the mouse brain by real-time quantitative polymerase chain reaction, using splice variant-specific primers, and western blot analysis. At the mRNA level,β-pro-GDNF expression was significantly greater than that of α-pro-GDNF in the mouse brain. In contrast, at the protein level,α-pro-GDNF expression was markedly greater than that of β-pro-GDNF. To clarify the mechanism underlying this inverse relationship in mRNA and protein expression levels of the GDNF splice variants, we analyzed the expression of sorting protein-related receptor with A-type repeats(SorLA) by real-time quantitative polymerase chain reaction. At the mRNA level, SorLA was positively associated with β-pro-GDNF expression, but not with α-pro-GDNF expression. This suggests that the differential expression of α-and β-pro-GDNF in the mouse brain is related to SorLA expression. As a sorting protein, SorLA could contribute to the inverse relationship among the mRNA and protein levels of the GDNF isoforms. This study was approved by the Animal Ethics Committee of Xuzhou Medical University, China on July 14, 2016.

Expression and significance of GDNF gene in recovery of spermatogenesis in mice

Objectives: To analyze the expression and significance of glial cell derived neurotrophic factor (GDNF) in the recovery of spermato genesis in mice. Methods: Adult Kunming mice were injected in traperitoneally with 2 doses of busulfan (10 mg/kg) 24 days apart so as to establish the spermatogenesis recovery modei. Testes were harvested at weeks l, 2, 3,4, 6, 8 and 10 after the second injection. Eight normai mice served as the controls. Recovery of spermatoge nesis was observed by light and electron microscopy and the GDNF mRNA measured by semi-quantitative RT-PCR and in situ hybridization. Results: After the second injection the expression of GDNF mRNA was increased significantly at week l and reached its peak at week 2. It was then decreased significantly at week 3 and reached its valley at week 4. After that it was increased gradu ally and recovered at week 10. GDNF mRNA was mainly ex pressed by the Sertoli celis. Conclusion: In the course of recovery of spermatogenesis, a high Ievel of GDNF expression plays a key role in the promotion of self-renewal and maintenance of the num ber of spermatogonial stem celis.

周围神经损伤后外源性GDNF对神经元的保护作用

采用硅管套接大鼠切断的坐骨神经模型 ,局部给予胶质细胞源性神经营养因子 (GDNF) ,应用尼氏染色、酶组织化学染色方法 ,观察到外源性GDNF能减少脊髓修复侧前角运动神经元死亡的数目 ,降低脊髓前角运动神经元及脊神经节感觉神经元中胆碱酯酶 (CHE)及酸性磷酸酶 (ACP)变化的幅度。这表明外源性GDNF能保护周围神经切断后引起的神经元损伤。

移植GDNF基因修饰的神经干细胞对大鼠脑卒中的神经保护作用

目的：探讨移植胶质细胞源性神经营养因子（GDNF）基因修饰的神经干细胞（NSCs）是否比单纯NSCs移植对脑卒中有更好的神经保护。方法：用GDNF重组质粒转染NSCs，构建过表达GDNF的NSCs（GDNF／NSCs）。插线法制备大鼠脑缺血卒中模型，再灌注3d，脑立体定位分别移植NSCs、GDNF／NSCs以及生理盐水到同侧侧脑室，实验分为GDNF／NSCs组、NSCs组和对照组。再灌注第1、2、3、5和7周处死大鼠，用改良的神经功能缺失评分和H-E染色分别评估大鼠神经功能和脑梗死体积；免疫组织化学显色观察移植的NSCs数量与分布和突触蛋白Syn、PSD-95在脑组织的表达。结果：再灌注2、3周，GDNF／NSCs组较NSCs组神经功能恢复更好。在各时间点GDNF／NSCs和NSCs组大鼠脑缺血体积较对照组显著减小；GDNF／NSCs组的NSCs细胞数量较NSCs组显著增多。再灌注2和3周，GDNF／NSCs组Syn免疫阳性产物比NSCs组显著增加；各时间点GDNF／NSCs组的PSD-95免疫阳性产物比NSCs组显著增加。结论：GDNF基因修饰的NSCs比NSCs对大鼠脑卒中有更好的神经保护作用。

GDNF重组腺病毒的构建及促进多巴胺能神经元存活的研究

利用体内同源重组原理，构建了能介导ＧＤＮＦ基因转移和表达的复制缺陷型重组腺病毒ＡｄＣＭＶｇｄｎｆ，其中ＧＤＮＦｃＤＮＡ插入腺病毒基因组的Ｅ１区并由ＣＭＶ启动子控制在人２９３细胞内通过同源重组包装生成重组腺病毒后，用形态学方法、病毒ＤＮＡ酶切分析、ＰＣＲ和ＲＴ－ＰＣＲ等方法进行鉴定正确．经测定病毒滴度达到１０１０ｐｆｕ／ｍｌ．用免疫沉淀方法从重组腺病毒感染的２９３细胞及其培养基上清中均检测到大量ＧＤＮＦ蛋白．用重组腺病毒直接感染或者用其条件培养基处理，分别使胚胎大鼠中脑原代多巴胺能神经元的数目增加８８．２％和９６．４％，明显增加多巴胺能神经元存活。

GDNF基因体内转染对大鼠脊髓损伤后轴突再生的影响

目的 :研究脂质体介导的胶质细胞源性神经营养因子 (GDNF)基因在大鼠损伤脊髓内的表达 ,观察外源性 GDNF对损伤脊髓轴突再生的作用。方法 :利用 Nystrom法制备大鼠胸髓压迫损伤模型。以直接注射法将脂质体 DC- Chol和重组质粒p EGFP- GDNF c DNA混合后注入大鼠损伤脊髓。采用 RT- PCR技术和荧光显微镜检测 GDNF基因转染后的体内表达 ,并通过辣根过氧化酶 (HRP)顺行追踪技术和神经微丝 (NF)、胶质原纤维酸性蛋白 (GFAP)免疫组化活性的变化来评价 GDNF基因转染对轴突再生的影响。结果 :经脂质体 DC- Chol介导 GDNF基因可有效地转染脊髓组织并得到表达。GDNF转基因 4周后可明显增加 NF阳性轴突数目 ,促进皮质脊髓束再生并通过损伤区。结论 :外源性 GDNF在损伤区局部高表达具有神经损伤保护作用 ,提示阳离子脂质体介导 GDNF体内转基因治疗创伤性脊髓损伤的方法是可行的。

嗅鞘细胞移植联合应用GDNF对大鼠脊髓损伤的治疗作用

目的 :研究嗅鞘细胞 (OECs)移植并联合应用胶质细胞源性神经营养因子 (GDNF)对脊髓损伤的保护和促进再生作用。方法 :建立成年 SD大鼠脊髓 T8半横断损伤模型 ,将体外培养纯化的 OECs植入脊髓损伤处 ,同时局部应用 GDNF。采用斜板试验和 BBB联合功能评分观察大鼠运动功能恢复情况 ,并通过辣根过氧化物酶逆行示踪技术评价 OECs和 GDNF对神经元存活和纤维再生的影响。结果 :(1) OECs和 GDNF联合应用时对皮质和红核神经元有逆行性保护作用 ,可促进脊髓下行传导束再生 ,肢体运动功能恢复。 (2 ) OECs和 GDNF联合应用组对脊髓损伤的修复作用最强 ,高于 GDNF或 OECs单用组。结论 :联合应用 GDNF和 OECs在脊髓损伤修复治疗中具有协同作用。

GDNF及HSV-GDNF对体外培养大鼠脊髓运动神经元受损后凋亡的影响

目的 观察胶质细胞源性神经营养因子 (GDNF)及单纯疱疹病毒载体介导的 GDNF(HSV- GDNF)对体外培养的胎鼠脊髓运动神经元在划痕损伤后凋亡的影响。 方法 对培养 12 d神经元行划痕损伤 ,并将其分成 4组 (无血清对照组、血清组、HSV- GDNF组和 GDNF组 ) ,给予不同培养液 ,定期观察各组的运动神经元存活数。分别于划痕损伤第 4d和第 7d时对神经元作 TU NEL 染色 ,检测运动神经元凋亡数 ,并在图像分析仪上对凋亡神经元作平均光密度的色谱分析。 结果 各组内运动神经元存活数与培养时间成反比。从对照组、HSV- GDNF组到 GDNF组 ,运动神经元凋亡数和凋亡神经元的平均光密度均依次减少 ,但对照组和血清组、GDNF组和 HSV-GDNF组间的运动神经元凋亡数以及凋亡神经元平均光密度均无显著差异。 结论 GDNF和 HSV- GDNF能挽救生长发育过程中受损的脊髓运动神经元的凋亡 ,对体外培养的受损脊髓运动神经元具有一定的保护作用。

GDNF重组腺病毒增加MN9D细胞多巴胺合成与释放

有表达ＧＤＮＦ的重组腺病毒直接感染中脑多巴胺能神经元来源的ＭＮ９Ｄ细胞并经神经毒素ＭＰＰ＋处理．感染３６ｈ分别收获细胞及其培养基，用反相高效液相色谱方法测定多巴胺含量．结果显示，经ＧＤＮＦ重组腺病毒感染的ＭＮ９Ｄ细胞内及其培养基中的多巴胺水平分别增加５０．７％和５３．５％．给予神经毒素ＭＰＰ＋损伤后，细胞内多巴胺含量降低５３．５％，但同时给予ＧＤＮＦ腺病毒则可抑制这种降低，并使多巴胺水平增加１４１．３％．以上结果提示ＧＤＮＦ腺病毒可提高细胞内的多巴胺水平并促进其释放，同时还具有对抗神经毒素ＭＰＰ＋损伤作用，表明ＧＤＮＦ重组腺病毒用于帕金森氏病基因治疗具有良好的前景．

GDNF及HSV-GDNF对坐骨神经损伤大鼠脊髓运动神经元Bcl-2表达的影响

目的 观察胶质细胞源性神经营养因子 (GDNF)及单纯疱疹病毒 (HSV)载体介导的 GDNF(HSV-GDNF)对坐骨神经损伤大鼠脊髓运动神经元 Bcl- 2表达的影响。 方法 分别取坐骨神经损伤后 4d、7d和 14d大鼠(分成对照组、GDNF组和 HSV- GDNF组 )的腰段脊髓 (L4～ 6 ) ,行石蜡包埋、切片 ;用抗 Bcl- 2抗血清进行免疫组织化学染色 ,观察 Bcl- 2免疫反应 (Bcl- 2 - IR)神经元数目 ,并在图像分析仪上对 Bcl- 2 - IR阳性神经元作光密度的色谱分析。 结果 1.坐骨神经损伤后 4d、7d时 ,GDNF组和 HSV- GDNF组损伤侧脊髓运动神经元 Bcl- 2 - IR阳性神经元的数量和平均光密度均明显高于对照组损伤侧。2 .坐骨神经损伤后 14d时 ,对照组、GDNF组和 HSV- GDNF组损伤侧脊髓运动神经元对 Bcl- 2的表达已无明显差别。 结论 GDNF与 HSV- GDNF能够增强坐骨神经损伤大鼠脊髓运动神经元 Bcl- 2的表达 ,减少神经元的退化死亡。

逆转录病毒载体ex vivo途径表达TH和GDNF基因

为了通过逆转录病毒载体 ex vivo途径高效表达 TH和 GDNF基因。本实验构建有 TH和 GDNF基因的重组逆转录病毒载体 p LHCX/TH和 p L NCX2 /GDNF,分别转染包装细胞 PA3 17和 PT67中 ;经筛选、RT-PCR、免疫组化等鉴定得到产毒细胞 PA3 17/TH和 PT67/GDNF。培养 COS-7细胞 ,以两种产毒细胞的上清分别感染 COS-7细胞 ,筛选后 ,免疫组化、原位杂交检测基因的表达量 ,将基因改造的细胞移植到大鼠纹状体内 ,免疫组化检测体内移植的 TH、GDNF基因的表达。结果表明 :TH和GDNF基因可在靶细胞表达 ,且筛选后基因表达阳性细胞显著增加 ;TH阳性率达 5 0 % ,GDNF阳性率达 70 %。在体内实验中 ,可以观察到这两种外源基因可同时在大鼠脑内移植区表达。以上结果提示 ,TH和 GDNF基因改造细胞 ,可通过 ex vivo途径在脑内移植区高效表达。这一实验结果将对

GDNF对脊髓前角运动神经元的保护作用

目的 :证实胶质源性神经营养因子 (glialcellline derivedneurotrophicfactor ,GDNF)对脊髓前角运动神经元有保护作用。方法 :切断SD大鼠一侧坐骨神经建立脊髓前角运动神经元损伤模型 ,借助单盲端硅胶管系统在损伤神经局部给予GDNF ,术后不同时间分别取L5脊髓切片 ,利用酶组织化学染色方法显示胆碱酯酶 (CHE)和酸性磷酸酶 (ACP)活性并进行图像分析。结果 :坐骨神经切断可导致腰段脊髓前角运动神经元明显损害 ,表现为CHE活性降低 ,ACP活性升高 ;应用GDNF可显著改善上述酶学变化。结论

大鼠坐骨神经切断后GDNF mRNA在其向心端及脊髓表达的变化

目的 :探讨大鼠坐骨神经切断后胶质细胞源性神经营养因子 (GDNF) m RNA在其向心端及脊髓表达的变化及其意义。 方法 :在切断 SD大鼠双侧坐骨神经后的不同时间采用半定量 RT- PCR方法 ,观察坐骨神经向心端及 T1 2 ～ L1 段脊髓GDNF m RNA表达的变化。 结果 :坐骨神经切断前 ,GDNF m RNA在坐骨神经及 T1 2 ～ L1 段脊髓微量表达 ,切断后其向心端及 T1 2 ～ L1 段脊髓表达逐渐减少 ,伤后 1、7、14、2 8d分别减少 10 %、38%、4 5 %、5 2 %及 2 0 %、6 8%、80 %、85 %。 结论 :坐骨神经切断后其向心端及 T1 2 ～ L1 脊髓 GDNF m RNA表达减少 ,推测是由于失去靶组织转运 GDNF m RNA所造成 ,为外源性GDNF应用于治疗脊髓损伤提供了实验依据。

大鼠GDNF基因的克隆表达及对PC12工程细胞的影响

克隆与表达大鼠胶质细胞源性神经营养因子 (glialcellline derivedneurotrophicfactor,GDNF)并观察其对PC12工程细胞的影响。从新生 4天的SD大鼠海马组织中提取总RNA ,通过RT PCR方法 ,扩增出GDNFcDNA。构建表达质粒pET GDNF ,转化大肠杆菌BL2 1(DE3) ,IPTG诱导GDNF表达并在Ni2 + NTA柱上用一步复性法纯化和复性。把PCDNA3 0 GFRα1和pcDNA3 0 RET质粒双转染入PC12细胞 ,G4 18筛选稳定克隆 ,构建PC12工程细胞。用GDNF刺激工程细胞 ,3天后观察其存活和分化。大鼠GDNFcDNA的克隆与表达获得成功 ,纯化和复性的重组GDNF蛋白可显著增强PC12 GFRα1 RET工程细胞的存活和分化。初步明确GDNF诱导PC12工程细胞存活和分化是通过RET 依赖途径。

GDNF促进帕金森病大鼠模型脑内移植的中脑源神经干细胞表达Pitx3、Nurr1和TH

目的:探讨胶质细胞源性神经营养因子(GDNF)作用下,中脑源神经干细胞(mNSCs)在帕金森病(PD)大鼠模型纹状体移植区表达垂体同源盒家族因子3(Pitx3)、孤儿核受体相关因子1(Nurr1)和酪氨酸羟化酶(TH)的变化。方法:mNSCs单独移植组、mNSCs+GDNF移植组和生理盐水假手术组的PD大鼠模型,移植4周和8周后采用免疫荧光和Western Blot方法检测移植区Pitx3、Nurr1和TH的表达。结果:(1)mNSCs单独移植组和mNSCs联合GDNF移植治疗组在移植后4周、8周移植区均有Pitx3,Nurr1和TH表达,mNSCs单独移植组较少表达Pitx3,Nurr1;(2)Western Blot检测显示联合移植组移植区Pitx3、Nurr1和TH表达量高于单独移植组,其中联合移植组4周表达量最高(P<0.05)。结论:GDNF有促进PD大鼠模型脑内移植的mNSCs表达Pitx3、Nurr1和TH的作用。

联合应用NGF、CNTF和GDNF对大鼠坐骨神经结构和功能恢复的影响

目的:探讨联合应用NGF、CNTF和GDNF对大鼠坐骨神经结构和功能恢复的影响。方法:采用大鼠坐骨神经离断模型,实验动物按NGF、CNTF、GDNF单独使用,两两组合使用,三种因子同时应用以及对照组共分为8组。测量各组坐骨神经功能指数、神经电生理参数、腓肠肌湿重恢复率、再生神经纤维形态参数。结果:三种因子联合治疗组坐骨神经功能指数、神经传导速度、动作电位波幅、腓肠肌湿重恢复最佳;除髓鞘厚度略小于NGF和CNTF联合治疗组外,神经纤维直径、再生轴突数量及神经组织面积均大于其他各组。结论:联合应用NGF、CNTF和GDNF对再生神经结构和功能恢复的作用优于其中一种因子单独使用或两种因子联合应用。

GDNF基因逆转录病毒表达载体的构建及转染神经干细胞的研究

应用基因重组技术将大鼠GDNF cDNA克隆到逆转录病毒载体pLXSN中,经酶切及PCR对重组质粒进行鉴定。将重组体包装到PA317细胞中,并感染增殖旺盛的大鼠神经干细胞,通过免疫细胞化学、RT-PCR和western-blot等方法鉴定转染效率。结果显示GDNF cDNA正确地克隆到逆转录病毒载体pLXSN中,该真核细胞表达载体成功转染增殖旺盛的神经干细胞并有效表达。因此本研究成功构建了GDNF真核细胞表达载体,并成功转染到神经干细胞中。为移植替代和基因治疗神经系统疾病提供了实验基础。