帕金森病基因治疗的实验研究

帕金森病(PD)的主要病理特征是中脑黑质多巴胺能神经元变性,表达的酪氨酸羟化酶(TH)减少或者活性降低。目前外源性多巴是最有效的抗PD药物,但常在数年后失去其有效的治疗作用。用胚胎脑细胞移植虽有效果,但胚胎脑来源困难。因此需要探索新的有效治疗方法。本研究将遗传修饰的成肌细胞植入偏侧PD鼠模型纹状体进行基因治疗。移植转TH基因的成肌细胞(治疗组,n=24)和未经遗传修饰的成肌细胞(对照组,n=10)于偏侧PD鼠损毁侧纹状体。用阿朴吗啡(APO)诱发旋转行为,RT-PCR、TH免疫组化检测TH基因的表达和TH蛋白的合成以及HPLC-ECD检测纹状体多巴胺及代谢产物含量以此评估基因治疗的效果。治疗组移植治疗后APO诱发的旋转行为明显改善(P<0.01),且可持续13个月,而对照组APO诱发的旋转行为无改善(P>0.05),应用RT-PCR、TH免疫组化和HPLC-ECD在治疗组移植部位检测到TH基因的表达、TH蛋白的合成和移植侧纹状体部多巴胺及其代谢产物含量增高。遗传修饰的成肌细胞能够在体内长时间、有效地表达TH,并改善PD鼠的病理行为,是PD基因治疗的合适靶细胞之一。     

Human myoblast transplantation: preliminary results of 4 cases.

Myoblasts from immunocompatible donors have been transplanted into the muscles (tibialis anterior, biceps brachii, and/or extensor carpi radialis longus) of 4 Duchenne patients in the advanced stages of the disease. Although no immunosuppressive treatment was used, none of the patients showed any clinical signs of rejection such as fever, redness, and inflammation. One patient transiently produced antibodies against the donor myoblasts as determined by cytofluorometric analysis. This patient and 2 others were shown to form antibodies against their donor's myotubes. Muscle biopsies of the injected tibialis anterior of 4 patients revealed that 80%, 75%, 25%, and 0% of the muscle fibers, respectively, showed some degree of dystrophin immunostaining. The contralateral noninjected muscles of the latter 3 patients did not contain any dystrophin positive fibers, while that of the first patient showed dystrophin expression in 16% of the fibers examined. Myoblasts were also injected into the extensor carpi radialis longus or the biceps brachii of these patients. A few months subsequent to injection, one patient was shown to have a 143% increase of strength during static wrist extension. This result must be interpreted with caution because a double-blind strength-measuring protocol was not used. Furthermore, we have noted that this change slowly decayed over time. The strength of 2 other patients was increased less remarkably (41% and 51%), while the strength of the fourth patient was unchanged.     

High efficiency transfection of primary skeletal muscle cells with lipid-based reagents

Lipofection is a convenient method for gene transfer into muscle cells but reportedly is inefficient. We tested the efficacy of commercially available lipid-based and polyamine transfection reagents. Primary rat skeletal muscle cell cultures were transfected at three stages of development and assayed after fusion. Efficiency reached 30% during the proliferation stage and up to 23% when most myoblasts had fused into myotubes. Optimization of transfection conditions with three different vectors yielded efficiencies exceeding 50%. Thus, lipid-based transfection into primary skeletal muscle cells can be several times more efficient than previously reported.     

Leukemia inhibitory factor enhances regeneration in skeletal muscles after myoblast transplantation

Cell-based therapies, such as myoblast transfer therapy, are likely to become an integral part of any approach to treat myopathies such as Duchenne muscular dystrophy. Previous studies have shown that an increased level of regeneration in the host muscle enhances incorporation of donor myoblasts. Leukemia inhibitory factor (LIF) increases the number of dystrophic fibers expressing dystrophin after myoblast transplantation and enhances regeneration in injured and diseased muscle. Morphometric analysis was used to investigate whether an increased level of regeneration is induced by LIF after myoblast transplantation. We found that, in muscles treated with LIF, the number of fibers undergoing regeneration was increased. The increased incorporation of donor myoblasts and thus dystrophin expression induced by LIF may be due, at least in part, to an increased level of regeneration of dystrophic muscle.     

探讨生肌调节因子和连接蛋白43基因在大鼠成纤维细胞中的表达

目的 通过观察转染生肌调节因子（myoblast determination，MyoD）基因和连接蛋白43（Connexin）基因的大鼠真皮成纤维细胞（demml fibroblast，DFs）生物学功能的变化，探讨Myo和Cx43基因转染DFs细胞的意义。方法采用Gateway技术，构建真核质粒表达载体，利用慢病毒（LV）表达系统，将大鼠MyoD cDNA和Cx43 cDNA转入大鼠成纤维细胞中，经Blasticidin筛选培养，通过RT-PCR，Western blot及膜片钳技术等方法检测MyoD及Cx43 mRNA及蛋白表达，并检测离子电流变化，通过显微镜观察转染后生长情况，免疫组织化学检测肌动蛋白和结蛋白的表达。结果RT-PCR，Westem blot检测出MyoD及Cx43的mRNA及相应蛋白表达，膜片钳检测到转染后钙离子电流，显微镜观察到基因转染筛选后，培养1周细胞的融合现象，并有多核肌管形成。免疫组织化学检测可见其下游蛋白Desmin及α-actin呈现阳性表达。结论MyoD和Cx43基因转染使DFs分化为成肌细胞，为进一步研究基因治疗心力衰竭奠定基础。     

成年大鼠成肌细胞的原代培养

目的　探索成年大鼠成肌细胞的原代培养方法。方法　取成年SD大鼠的胸大肌 ,利用组织块培养法培养成肌细胞。绘制生长曲线 ,并对培养细胞进行形态学研究和免疫细胞化学鉴定。结果　采用组织块培养法 ,2周后成肌细胞的密度可达 10 7/ml,免疫细胞化学分析显示 90 %以上的细胞呈骨骼肌特异的肌细胞生成素 (myogenin)抗体染色阳性。结论　利用组织块法成功培养成年大鼠原代成肌细胞 ,该方法实用性强 ,所得成肌细胞纯度高 ,为深入研究心肌梗死疤痕中成肌细胞自体移植奠定了基础。     

Rapid death of injected myoblasts in myoblast transfer therapy

Myoblast transplantation has been proposed as a potential therapy for Duchenne muscular dystrophy (DMD). A Y-chromosome-specific probe was used to track the fate of donor male myoblasts injected into dystrophic muscles of female mdx mice (which are an animal model for DMD). In situ analysis with the Y-probe showed extremely poor survival of isolated normal male (C57B1/10Sn) donor myoblasts after injection into injured or uninjured muscles of dystrophic (mdx) and normal (C57B1/10Sn) female host mice. A decrease in the numbers of donor (male) myoblasts was seen from 2 days and was marked by 7 days after injection: few or no donor myoblasts were detected in host muscles examined at 3–12 months. There was limited movement of the injected donor myoblasts and fusion into host myofibers was rare. The results of this study strongly suggest that the failure of clinical trials of myoblast transplantation therapy in boys with DMD may have been due to rapid and massive death of the donor myoblasts soon after myoblast injection. © 1996 John Wiley & Sons, Inc.     

NO抑制体外IFN-γ刺激成肌细胞/肌管NLRP3炎性小体活化

目的观察一氧化氮供体硝普钠(SNP)、一氧化氮合酶抑制剂(L-NAME)对体外炎性培养(IFN-γ刺激)成肌细胞/肌管NLRP3炎症小体活化的影响。方法利用IFN-γ刺激小鼠C2C12成肌细胞/肌管,q PCR和Western blot分析炎性小体(ASC、NLRP3、caspase-1)的形成及活化、ELISA检测细胞培养上清IL-1β的分泌。进一步利用L-NAME和SNP处理IFN-γ诱导的C2C12细胞/肌管,分析炎性小体的形成、活化及IL-1β的分泌。结果 IFN-γ刺激培养后,成肌细胞/肌管中NLRP3、ASC和mature-caspase-1表达水平上调(P0.01)。较之未刺激的细胞,IFN-γ诱导会显著上调成肌细胞/肌管培养基中的IL-1β浓度(P0.01)。SNP处理后6 h,分化肌管(IFN-γ刺激48 h)内炎性小体(ASC、NLRP3、caspase-1)mRNA和蛋白水平较单纯刺激细胞显著下调(P0.01),L-NAME则上调ASC、NLRP3、caspase-1表达水平(P0.05)。与上述结果一致,SNP和L-NAME处理同时分别下调或上调培养基中IL-1β浓度(P0.05)。结论在炎性条件下,成肌细胞/肌管具备合成并活化NLRP3炎性小体的能力。NO对炎性小体的形成及活化有一定的抑制作用。     

周期性牵张面颌致成肌细胞内Ca2+浓度变化的研究

目的观察牵张引起的成肌细胞内Ca2+浓度变化,探求口腔正畸功能矫形治疗中肌肉改建机制。方法采用四点弯曲加力装置,使体外培养的SD大鼠面颌部成肌细胞发生牵张变形后,测定加力后不同时段及在镜下给予乙酰胆碱(Ach)后成肌细胞内Ca2+荧光强度的变化。结果牵张引起的成肌细胞内Ca2+荧光强度的升高明显高于Ach的作用,并且在经历了体外的牵张加力后,Ach刺激并未使成肌细胞内Ca2+荧光强度出现预先设想的叠加效果,在各个不同的实验组中Ca2+荧光强度反而略有下降。结论牵张引起的骨骼肌细胞内Ca2+浓度升高的机制不同于肌肉兴奋收缩耦联过程中出现的Ca2+浓度升高,Ca2+作为第二信使引发的细胞内各种结构和功能蛋白的改变可能参与了肌肉改建过程。