目的 利用原位杂交检测味细胞中G蛋白的表达。方法 分别用RT-PCR方法克隆大鼠味蛋白及（a-rod）转导蛋白的全长cDNA，制备地高辛标记的正义链及反义链探针。结果原位杂交分析了150个大鼠味蕾，结果显示味蛋白和转导蛋白在舌味蕾细胞中都有表达。但两种蛋白标记细胞数量有明显差别。每个味蕾大约有1.2个转导蛋白阳性细胞，占总味蕾细胞的0.8%；而每个味蕾平均有6.2个味蛋白阳性细胞，占总味蕾细胞的4.1%；即味蕾中味蛋白阳性细胞大约是转导蛋白阳性细胞的5倍。转导蛋白探针对味蛋白mRNA无交叉杂交反应。在视网膜上可见转导蛋白基因的高度表达，而无味蛋白的表达。结论味蛋白和转导蛋白可能是味细胞的G-蛋白，但以味蛋白特异性表达为主。它们可能参与味觉的信号转导作用。

目的 研究人三磷酸鸟苷酸环化水解酶Ⅰ（GCH Ⅰ）基因在多巴胺代谢过程中的作用。方法 从人胚胎肝脏中提取总RNA，以RT－PCR法扩增GCH ⅠcDNA，克隆于PGEM－T－easy载体中，测序正确后+G18再构建真核表达载体，转染猴肾成纤维细胞系COS7，原位杂交检测其表达。结果 RT-PCR扩增出904bp的cDNA，并成功构建真核表达载体pCI-neo-GCH Ⅰ，原位杂交证实其在COS7表达阳性率为70%～80%。结论 GCH Ⅰ有望用于帕金森病的基因治疗。

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pLHCX/TH和pLNCX2/GDN F，分别转染包装细胞PA317和PT67中；经筛选、RT-PCR、免疫组化等鉴定得到产毒细胞PA 317/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50%，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Park inson病复合性基因治疗提供依据。

目的 利用脑型及味型mGluR4在体内外的表达，检测两种受体结构功能上的差异，从而证实它们在信号转导功能上的不同。方法 将克隆得到的脑型及味型mGluR4分别通过脂质体介导转染CHO 细胞。在L-MSG和L-AP4 的作用下，检测两种受体的功能。利用Western Blot及原位杂交技术，分别检测它们在体内外的表达。结果 转染脑型及味型mGluR4的CHO细胞，表达两种不同分子量的免疫活性蛋白，其中脑型分子量约为120kD而味型约为68kD。在L-MSG和L-AP4作用下，味型mGluR4对刺激物反应的浓度远高于脑型mGluR4。对大鼠脑组织及舌味蕾的原位杂交证明，mGluR4 在小脑颗粒细胞有很高的表达，而在味蕾细胞表达率较低，阳性细胞仅占味蕾细胞的5%。结论 味组织中的mGluR4 与脑组织中mGluR4在结构和功能上有明显不同。味型mGluR4特异性表达于味蕾细胞，在高于脑内的浓度下，结合细胞外的谷氨酸，通过降低cAMP的浓度引发味觉信号转导。是谷氨酸味觉的特异性受体。

目的 是探索骨髓基质细胞的分离培养、鉴定及其接受并表达TH基因的能力。实验中通过密度梯度离心法成功地从成年SD大鼠骨髓中分离获得了骨髓基质干细胞，并用流式细胞仪对其进行鉴定，纯度可达75%。进一步采用复制缺陷型腺相关病毒载体介导的基因转染方法, 将之改造成为携带lacZ与TH基因的工程细胞，经X-gal 染色和TH免疫组化检测，转染效率为（74.6±19.4）%。实验结果表明骨髓基质细胞易于接受并表达外源基因，有望作为运载细胞应用于帕金森病的基因治疗。

Receptor proteins for photoreception have been studied for several decades. More recently, putative receptors for olfaction have been isolated and characterized. In contrast, no receptors for taste have been identified yet by molecular cloning. This report describes experiments aimed at identifying a receptor responsible for the taste of monosodium glutamate (MSG). Using reverse transcriptase (RT)-PCR, we found that several ionotropic glutamate receptors are present in rat lingual tissues. However, these receptors also could be detected in lingual tissue devoid of taste buds. On the other hand, RT-PCR and RNase protection assays indicated that a G-protein-coupled metabotropic glutamate receptor, mGluR4, also is expressed in lingual tissues and is limited only to taste buds. In situ hybridization demonstrated that mGluR4 is detectable in 40-70% of vallate and foliate taste buds but not in surrounding nonsensory epithelium, confirming the localization of this metabotropic receptor to gustatory cells. Expression of mGluR4 in taste buds is higher in preweaning rats compared with adult rats. This may correspond to the known higher sensitivity to the taste of MSG in juvenile rodents. Finally, behavioral studies have indicated that MSG and L-2-amino-4-phosphonobutyrate (L-AP4), a ligand for mGluR4, elicit similar tastes in rats. We conclude that mGluR4 may be a chemosensory receptor responsible, in part, for the taste of MSG.

为了构建α-synuclein-pEGFP真核表达载体，检测其在SH-SY5Y细胞内的表达，本研究应用下述方法：PCR扩增α- synuclein基因并消除终止密码子；PCR产物连入pGEM T-easy载体，经测序确认无误后，亚克隆入pEGFP-N1，构建α-synucle-in-pEGFP真核表达载体；LipofectAMINE法转染SH-SY5Y细胞；荧光显微镜检测报告基因表达产物EGFP，原位杂交和免疫荧光细胞化学法检测α-synucleinmRNA及其蛋白表达。结果显示，该载体转染SH-SY5Y细胞后，可在细胞内观察到报告基因和目的基因的表达产物。结论：α-synuclein-pEGFP真核表达载体构建成功，并可在细胞内表达。本工作为今后动态观察研究α-synu-clein致Parkinson病多巴胺能神经细胞损伤机制奠定基础。

We have shown previously that in vitro ischemia could induce apoptosis in primary culture of astrocytes. In this paper we demonstrate that astrocytes in culture could undergo apoptosis during in vitro incubation postischemia. We also measured the changes of phosphorylated Erk1/2 (p-Erk1/2) and phosphorylated Akt (p-Akt) in order to determine whether these two pathways play a role in apoptosis. After 4 h in vitro ischemic incubation of cultured astrocytes, a limited amount of nuclear condensation was demonstrated by Hoechst 33342 staining. When ischemic incubation was halted and the cultures transferred to standard normoxic incubation (postischemia) conditions, DNA fragmentation and apoptosis were demonstrated by TUNEL and DNA laddering analysis. TUNEL-positive astrocytes began to appear at 6 h postischemia and increased in number from 12 h postischemia. Western blot analysis showed that both p-Erk1/2 and p-Akt were elevated in astrocytes subjected to 4 h of ischemia. Elevated p-Erk1/2 levels were sustained during the postischemia incubation for 12 h and decreased significantly afterward, but did not return to the levels in the control cultures that did not experience ischemic insult. In contrast, the p-Akt level continued to increase at 6 and 12 h postischemia before declining significantly. The changes in p-Erk1/2 and p-Akt correlated well with the appearance of apoptotic astrocytes in the culture.

Substantial evidence has shown that extracellular signal-regulated kinases 1 and 2 (Erk1/2) and serine/threonine kinase (Akt) play important roles in regulating cell survival. We examined the activities of these kinases in astrocytes under ischemia in an anaerobic chamber. The level of phosphorylated Erk1/2 in astrocytes began to increase after 1 h ischemia, reached a maximum after 4 h ischemia, before decreasing from 5 to 6 h. Akt was activated later than Erk1/2. It was significantly increased after 4 h ischemia before declining steadily afterwards. Lactate dehydrogenase (LDH) assay and Hoechst nucleic staining indicated that U0126, which inhibits Erk1/2 phosphorylation, enhanced ischemia-induced cell death, whereas LY294002, which inhibits Akt phosphorylation, delayed cell death. These effects were dose-dependent. At 4 and 6 h ischemia, U0126-treated astrocytes expressed a lower level of Bcl-2 than controls. In contrast, LY294002-treated astrocytes expressed a higher level of Bcl-2 than controls as shown by Western blots. Bcl-xL expression level was not affected by either treatment. These data suggest that activation of the MAPK/Erk1/2 pathway might protect astrocytes from ischemic injury, but activation of the PI3-K/Akt pathway does not. The effect may involve Bcl-2 but not Bcl-xL expression.

In situ hybridization (ISH) using nonradioactive probes enables mRNAs to be detected with improved cell resolution but compromised sensitivity compared to ISH with radiolabeled probes. To detect rare mRNAs, we optimized several parameters for ISH using digoxygenin (DIG)-labeled probes, and adapted tyramide signal amplification (TSA) in combination with alkaline phosphatase (AP)-based visualization. This method, which we term TSA-AP, achieves the high sensitivity normally associated with radioactive probes but with the cell resolution of chromogenic ISH. Unlike published protocols, long RNA probes (up to 2.61KB) readily permeated cryosections and yielded stronger hybridization signals than hydrolyzed probes of equivalent complexity. RNase digestion after hybridization was unnecessary and led to a substantial loss of signal intensity without significantly reducing nonspecific background. Probe concentration was also a key parameter for improving signal-to-noise ratio in ISH. Using these optimized methods on rat taste tissue, we detected mRNA for mGluR4, a receptor, and transducin, a G-protein, both of which are expressed at very low abundance and are believed to be involved in chemosensory transduction. Because the effect of the tested parameters was similar for ISH on sections of brain and tongue, we believe that these methodological improvements for detecting rare mRNAs may be broadly applicable to other tissues.