淋巴瘤细胞转染GM-CSF 基因后生物学特性的变化

目的：制备高表达GM－CSF的淋巴瘤细胞系，观察淋巴瘤细胞转染GM－CSF基因后生物学特性的改变。方法：将小鼠GF－CSF真核表达质粒用电穿孔法导入小鼠淋巴瘤细胞系RMA，有限稀释法制备单个细胞克隆，经RT－PCR，骨髓祖细胞增殖实验和集落形成实验筛选相对高表达GM－CSF的RMA克隆，并观察其生物学特性的改变。结果：获得了高表达GM－CS的RMA细胞系,其同基因动物体内致瘤性降低。结论：淋巴瘤细胞系RMA转染GM－CSF基因后致瘤性降低。     

表皮生长因子对牛眼小梁细胞c—fos基因表达的诱导

目的研究表皮生长因子（ｅｐｉｄｅｒｍａｌｇｒｏｗｔｈｆａｃｔｏｒ，ＥＧＦ）对小梁细胞的作用。方法应用分子杂交技术研究ＥＧＦ对体外培养的牛眼小梁细胞ｃ－ｆｏｓ基因表达的诱导和３Ｈ胸腺核苷酸（３Ｈ－ｔｈｙｍｉｄｉｎｅｉｎｃｏｒｐａｒａｔｉｏｎ，３Ｈ－ＴｄＲ）掺入法观察细胞ＤＮＡ的合成。结果小梁细胞的３Ｈ－ＴｄＲ掺入率随着ＥＧＦ浓度不同而变化，浓度为２０～１５０ｎｇ／ｍｌ时，细胞掺入率随浓度增加升高（Ｐ＜０．０１）。与对照组相比，ＥＧＦ刺激停止生长的小梁细胞０．５小时后，ｃ－ｆｏｓ基因开始表达，１小时后达高峰，至２小时后消失。不同浓度ＥＧＦ刺激小梁细胞１小时后，ｃ－ｆｏｓ基因表达呈浓度依赖性。结论实验结果表明ＥＧＦ刺激小梁细胞增殖或进入生长状态，可能与ｃ－ｆｏｓ基因产物的信号传递作用有关。     

Detailed mapping of CGRP mRNA expression in the rat central nervous system: Comparison with previous immunocytochemical findings

The localization of CGRP mRNA in neurons of the rat brain and spinal cord was assessed by in situ hybridization histochemistry (ISH) using a radiolabeled synthetic 57-mer oiigodeoxynucleotide probe complementary to the rat prepro CGRP mRNA. Results were compared with previously published findings of CGRP-immunoreactive (CGRP-IR) cell bodies revealed by an indirect immunofluorescence technique. The highest numbers of CGRP mRNA expressing neurons as well as the greatest intensity of staining were found in the lateral hypothalamic area, the parabrachial nuclei, and among the cranial motor nuclei, especially in the nuclei of the 7th and 12th nerve and the ambiguus nucleus, which is generally in good agreement with findings assessed by immunocytochemistry (ICH). However, some mismatches between the localizaton of the peptide by ICH and the localization of the CGRP mRNA were also observed. Thus, ISH was not able to confirm CGRP-IR in cells of the amygdaloid complex and parts of the medial hypothalamus, the central gray, and the inferior colliculus, but ISH revealed considerably more CGRP mRNA expressing cells in the lateral hypothalamic area, arcuate nucleus, posterior and peripeduncular thalamic nuclei, and all cranial motor nuclei than CGRP-IR containing cells found by ICH. Moreover, ISH also revealed CGRP mRNA synthesis in the nucleus of the lateral olfactory tract and in the perihypoglossal nuclei that were devoid of CGRP-IR. The reasons for the observed mismatches still remain to be elucidated; however, intracerebroventricular colchicine pretreatment used to increase immunocytochemical signals also might have induced or suppressed gene expression in certain brain regions in an unpredictable matter. On the other hand, detection of only the mRNA in a certain region does not necessarily mean that also the active peptide is synthesized there.     

nm23-H1基因转染对人胆管癌细胞系QBC939体外浸润能力的影响

目的：探讨nm23-H1基因转染对人胆管癌细胞系QBC939体外浸润能力的影响。方法：将含有全长nm23-H1 cDNA的真核表达载体通过脂腩体法转染人胆管癌细胞系。结果：转染成功的QBC939细胞，其nm23-Hl基因的mRNA、蛋白表达明显增加，转染nm23-H1基因的胆管癌细胞体外浸润能力下降，穿越matrigel的细胞数明显低于亲本QBC939细胞，代表浸润能力的IV型胶原酶（MMP-9）分泌量下降。结论：nm23-Hl基因可以抑制胆管癌细胞的体外浸润能力。     

中国汉坦病毒H82O5株G2糖蛋白基因的克隆及在真核细胞中的瞬时表达

从感染病毒乳鼠脑组织提取总RNA，采用RT－PCR和分子克隆技术将扩增到的G2糖蛋白基因插入含CMV启动子的pcDNA3.1/His质粒载体中，通过脂质体介导转染COS－7细胞，用SDS－PAGE、Western-blot及IFIA方法分别测定表达产物的相对分子量及特异性。结果证明获得正向插入的G2－pcDNA3.1/His重组表达质粒，表达产物的相对分子量为56ku，与理论预期大小一致，并且可与汉坦病毒H8205株的腹水抗体起特异反应。表明构建的G2－pcDNA3.1/His重组质粒所表达的蛋白为中国汉坦病毒株特有，能在哺乳动物细胞中表达并具有抗原性，重组质粒可应用于汉坦病毒的DNA疫苗研究。     

白血病细胞p16基因突变分析

目的探讨ｐ１６基因突变在白血病发生中的作用及基因突变的机制。方法利用点突变检测仪、水平和垂直板电泳对ｐ１６基因的外显子１、外显子２的ＰＣＲ扩增产物作缺失和点突变分析。结果在白血病３５例临床标本中有２２例发生缺失突变，６例发生点突变，突变率约８０％。在２２例缺失突变病例中，有１０例为不完全缺失突变即有低于外显子５０９ｂｐ的扩增产物。结论ｐ１６基因含有“ＧＣ”ＤＮＡ重复顺序，易发生ＤＮＡ重组及易位和重排。在白血病发生中起重要作用     

人睫状神经营养因子结构和功能的研究

制备高活性的重组人睫状神经营养因子，并研究其生物学功能。方法：应用大肠杆菌表达ｈＣＮＴＦ，用片段插入和法研究其结构与功能关系；切断大鼠骨神经，局部及皮下给予ＣＮＴＦ，应用辣根过氧化物酶逆行追踪技术显示再生轴突通过修复部位的胞体。结果；ｈＣＮＴＦ分子中α－螺旋结构的维持对其生物活性十分重要Ｃ端松散地其生物活性贡献不大，Ｄ螺旋中后段可能与生物活性有密切关系；     

Effects of capsaicin in temporomandibular joint arthritis in rats

Temporomandibular joint (TMJ) arthritis was induced in female Lewis rats by unilateral injection of a suspension of heat-killed Mycobacterium butyricum in paraffin oil into the TMJ. Control rats received paraffin oil by the same route. Arthritic and control rats were pretreated either with capsaicin or denervation of the mandibular branch of the trigeminal nerve. Tissues were collected for neuropeptide extraction and analysed by radioimmunoassay and reverse-phase high-performance liquid chromatography. In all groups, the levels of substance P- (SP), calcitonin gene-related peptide- (CGRP) and neuropeptide Y- (NPY) like immunoreactivity (LI) were higher in the trigeminal ganglia than in the TMJs. In control rats, capsaicin significantly lowered the levels of SP-LI in the trigeminal ganglia and TMJ, but not CGRP-LI and NPY-LI. In the arthritic rats, capsaicin pretreatment significantly lowered the SP-LI and CGRP-LI in the trigeminal ganglia and TMJ, but not the NPY-LI. In the trigeminal ganglia the unilateral denervation significantly lowered SP-LI in control rats, and in arthritic rats SP-LI and CGRP-LI. On the denervated side of the arthritic TMJ, NPY-LI, SP-LI and CGRP- LI were significantly lowered as compared to the arthritic control rats and to the contralateral side. In this rat model, pretreatment with capsaicin and surgical denervation decreased the neuropeptide content in the trigeminal ganglia and the TMJ. The results clearly demonstrate a close interaction between increased neuropeptide release from sensory and sympathetic neurones after induction of arthritis in the rat.     

Synthesis of TPH-13, a new tridecapeptide from Phyllomedusa rohdei

The synthesis of TPH-13 (Glp-Glu-Lys-Pro-Tyr-Trp-Pro-Pro-Pro-Ile-Tyr-Pro-Met-OH), a tridecapeptide isolated from the skin of the South American frog Phyllomedusa rohdei, is described and alternative approaches are discussed.