脂质体介导外源基因体外转染精子的研究

为探索精子作为外源基因的载体建立转基因小鼠的可行性。方法我们从性成熟小鼠的输精管及部分附睾管中取出精子，与阳离子脂质体包裹的ＤＮＡ混合孵育之后将其注入超排卵小鼠的阴道内，４８ｈ后收集Ⅱ细胞胚胎细胞。结果（１）精子可转染上外源ＤＮＡ，转染率达５３％；（２）转染有外源ＤＮＡ的精子可使Ⅱ细胞期胚胎携带上外源基因，携带率为１１．５％。结论小鼠输精管及附睾管内的精子可被脂质体包裹的外源ＤＮＡ转染并可作为载体     

精子细胞输精管及附睾管内转染法建立转基因小鼠

目前，生产转基因动物主要的方法仍是受精卵显微注射法，所需设备复杂，技术难度大，成功率低。为此，人们都在寻找一种新的方法。利用精子携带外源DNA进入卵母细胞的精子载体法是设想之一。九十年代初，Mare和Rottmann等分别报导了电击法和脂质体转染法让精子在体外携带上外源DNA，再行体外受精的方法，引起了学者们的广泛关注，但其结果不稳定，阳性率极低。我们采用脂质体包裹外源DNA，直接注入输精管内，数天后与雌鼠交配的方法，得到了较为满意的结果，现简报如下。1材料和方法将构建好的WAP－t－PA质粒与脂质体混合制备成转染…     

精原干细胞体内转染途径建立rsP3111转基因小鼠的可行性研究

目的构建目的基因rsp3111与荧光蛋白融合表达的质粒,并且实现目的基因在体外受精胚胎及在体附睾成熟精子中表达。为进一步通过精原干细胞体内转染途径建rsp3111转基因小鼠奠定基础。方法将rsp3111基因以Xholl和BdmHI酶切后插入经同样酶切处理的真核细胞表达质粒pDsRed-Monomer—N1中,重组质粒为pDsRed-Monomer—N1-rSP3111。采用共培养和脂质体介导方法将ICR小鼠精子与pDsRed-Monomer—N1-rSP3111质粒共孵育,再与成熟的卵母细胞体外受精;或用脂质体介导睾丸直接注射法,将pDsRed-Monomer—N1-rSP3111导入小鼠睾丸中,通过PCR及荧光显微镜观察体外受精后的受精卵及体内睾丸转染rsp3111基因后附睾精子中是否有外源rsp3111基因的表达。结果PCR结果表明,rsp3111基因成功地转入了附睾精子及受精卵中。荧光检测结果表明,目的基因rsp3111在体外受精胚胎及睾丸的曲细精管中均有表达。结论通过雄性生殖细胞载体法实现了大鼠配子特异性蛋白rSP3111在小鼠受精卵及睾丸、附睾精子中的表选为进一步通过精原干细胞体内转染途径建立rsp3111转基因小鼠奠定了基础。     

低浓度臭氧对小鼠生长发育的影响

目的构建目的基因rsp3111与荧光蛋白融合表达的质粒,并且实现目的基因在体外受精胚胎及在体附睾成熟精子中表达。为进一步通过精原干细胞体内转染途径建rsp3111转基因小鼠奠定基础。方法将rsp3111基因以Xholl和BdmHI酶切后插入经同样酶切处理的真核细胞表达质粒pDsRed-Monomer—N1中,重组质粒为pDsRed-Monomer—N1-rSP3111。采用共培养和脂质体介导方法将ICR小鼠精子与pDsRed-Monomer—N1-rSP3111质粒共孵育,再与成熟的卵母细胞体外受精;或用脂质体介导睾丸直接注射法,将pDsRed-Monomer—N1-rSP3111导入小鼠睾丸中,通过PCR及荧光显微镜观察体外受精后的受精卵及体内睾丸转染rsp3111基因后附睾精子中是否有外源rsp3111基因的表达。结果PCR结果表明,rsp3111基因成功地转入了附睾精子及受精卵中。荧光检测结果表明,目的基因rsp3111在体外受精胚胎及睾丸的曲细精管中均有表达。结论通过雄性生殖细胞载体法实现了大鼠配子特异性蛋白rSP3111在小鼠受精卵及睾丸、附睾精子中的表选为进一步通过精原干细胞体内转染途径建立rsp3111转基因小鼠奠定了基础。     

阳离子脂质体介层的外源基因转染小鼠脾细胞的方法学探讨

目的：探讨高效阳离子脂质体介导外源基因转染体外生长的小鼠脾细胞的方法。方法：用新型阳离子脂质体DOSPER包裹携带lac Z报告基因的质粒pCAGGS-lacZ按3种方法转染小鼠脾细胞，（1）常规方法直接转染；（2）脾细胞经刀豆球蛋白A（ConA)活化后再按常规方法转染；（3）用黏附辅助脂质体法（adhesion-assisted lipofection,AAL）转染脾细胞。转染效率用X－gal染色法测定。结果：上述3种方法的转染效率依次为＜0．010，0．057及0．199，经方差分析，3种方法转染效率的差异有显著性（P＜0．01，n=3).结论：AAL法介导外源基因转染体外生长的小鼠脾细胞的转染效率最高。     

不同山羊个体的精子结合和内化外源DNA能力的差异性研究

目的探讨不同山羊个体的精子结合和内化外源DNA能力差异性，以及精子内化能力对制备转基因山羊胚胎阳性率的影响。方法用地高辛标记与检测试剂盒检测12只山羊的精子分别结合、内化质粒DNApEGFP-N1的效率；体外受精实验检测胚胎阳性率与精子内化pEGFP-N1能力的关系。结果不同山羊个体的精子结合和内化pEGFP-N1的效率存在差异，内化效率最高为53．7％，最低为3．1％；3号公羊的精子体外转染外源基因后行体外受精，所得的2～8细胞胚胎表达外源基因pEGFP-N1的阳性率最高，7、8、10、11号公羊的精子体外转染外源基因后行体外受精，所得的2～8细胞胚胎均未表达GFP蛋白。结论不同山羊个体的精子自发结合、内化外源DNA的能力存在差异；不同山羊个体的精子体外转染外源基因后行体外受精，所得的2～8细胞胚胎表达外源基因的比例不同。     

炎症诱导性表达载体的表达特性

目的：构建一种具有自控性的炎症诱导性表达载体，为探索符合临床实际的抗炎基因治疗新系统奠定基础，方法：（1）将对炎症敏感的SAA3启动子调控片优现报告基因CAT片段重组，构建炎症诱导性表达载体pSAA3/CAT;(2)用脂质体转染法将表达载体pSAA3/CAT转入体外培养细胞或动物体内，观察不同时间，不同剂量的炎性刺激时报告基因的表达变化，分析SAA3启动子体内外转录活性和炎症诱导性。结果：（1）阳离子脂质体包裹表达质粒可以有效地转染肝源性细胞SMMC－7721和单核细胞源性细胞U937，经活化后的U937细胞可迅速表达CAT，6h启动，24h达峰值，72h渐降至基础水平，（2）腹腔注射脂质体包裹DNA可有效转染外源报告基因CAT在体内表达；LPS活化的基因转染小鼠肝脏中的CAT表达与一次典型的急性反应时间一致，鼠肝CAT表达水平与LPS有显著的剂量依赖关系。结论：初步建立了一种具有自控性的有效抗炎基因治疗新系统。     

脂质体介导TGF-β1质粒DNA转染家兔角膜上皮细胞的实验研究

为证实TGF-β1质粒DNA能否由脂质体携带进入家兔角膜上皮细胞，用多聚阳离子脂质体携带重组的人TGF-β1质粒转染体外培养的家兔角膜上皮细胞，在转染12h后，表达2d时，用免疫组化方法（SABC）检测TGF-β1质粒DNA的表达情况，结果显示：外源TGF-β1质粒DNA在家兔角膜上皮细胞可以获得表达，基因转染率为23.37%。提示：脂质体作为角膜上皮细胞基因类药物介入的基础研究和应用研究的介导体具有广阔的应用前景。     

阳离子脂质体介导的外源基因转染小鼠脾细胞的方法学探讨

【目的】探讨高效阳离子脂质体介导外源基因转染体外生长的小鼠脾细胞的方法。【方法】用新型阳离子脂质体DOSPER包裹携带lacZ报告基因的质粒 pCAGGS lacZ按 3种方法转染小鼠脾细胞 ,①常规方法直接转染 ;②脾细胞经刀豆球蛋白A(ConA)活化后再按常规方法转染 ;③用黏附辅助脂质体法 (adhesion assistedlipofection ,AAL)转染脾细胞。转染效率用X gal染色法测定。【结果】上述 3种方法的转染效率依次为 <0 0 10 ,0 0 5 7及 0 199,经方差分析 ,3种方法转染效率的差异有显著性 (P <0 0 1,n =3)。【结论】AAL法介导外源基因转染体外生长的小鼠脾细胞的转染效率最高。     

包裹E1A基因的纳米粒子制备及转染人肺腺癌细胞A549实验研究

目的制备包裹E1A基因（腺病毒早期表达基因）的纳米粒子,并观察其介导E1A基因转染人肺腺癌细胞A549的可行性和效率。方法应用聚乳酸聚乙醇酸共聚物和聚乙烯醇包载E1A基因,制备纳米级粒子混合物,检测其包埋率、体外释放情况及粒径大小。用制备的包裹DNA纳米粒子转染人肺腺癌细胞A549,并以阳离子脂质体为对照,用PCR、RT-PCR方法分别检测转染细胞中E1A基因DNA整合和mRNA表达。结果制备的纳米粒子粒径为150～280nm,包埋率为0.78%,体外释放约为22d;在转染相等质量的DNA情况下,纳米组所得克隆数较脂质体组多（P〈0.05）;PCR、RT-PCR结果表明纳米粒子和脂质体转染细胞均有E1A基因整合和mRNA表达。结论成功制备了纳米粒子,纳米粒子可携带外源基因进行基因转染。     

不同浓度HFMC对大鼠输精管通畅性与生育可复性的影响

Great efforts have been made to develop non-occlusive or non-ever-lasting-occlusive contraception devices in vas deferens during the recent decade at home and abroad. In our previous studies both in vivo and in vitro, polymer HFMC was found to have a spermicidal effect. In this investigation in SD rats, the relationship between the concentration of HFMC injected into the vas deferens and the resumption of fertility was studied by means of continual observations on the existence of sperms in vaginal smears from the caged female and on pup-birth delaying, so as to determine occlusion of the vas lumen and the effect of HFMC on contraception in male. The histopathological changes in the vas deferens and caudal epididymis of the HFMC-injected male rats were also observed. The results indicated: (1) HFMC in concentration over 7.5% could show temporary contraception effect by its slow releasing of H+; (2) HFMC could induce definite contraception effect which lasted longer as the concentration of HFMC given increased; (3) HFMC had no effect on the descendents in number, mortality, sex ratio, body weight and appearance; (4) the bilateral internal reproductive structures were identified normal after resumption of fertility, but 20% HFMC induced vas deferens occlusion and spermatoceles; (5) moderate concentration of HFMC was considered favorable for reversible intra-vas deferens contraception.     

小鼠冷冻胚胎和精子SNP遗传鉴定方法的建立

【摘要】目的 建立小鼠冷冻胚胎和精子SNP(Single-Nucleotide Polymorphism)分型方法,用于冷冻胚胎和精子快速遗传鉴定方案。方法 本研究以中科院上海实验动物中心（国家啮齿类实验动物种子中心上海分中心）提供的小鼠冷冻胚胎和精子为样本,采用全基因组扩增技术和PCR-LDR分型技术建立小鼠冷冻物SNP遗传鉴定方法。结果 全基因组扩增技术能大幅度增加冷冻胚胎样本的DNA总量;PCR-LDR分型方法适用于小鼠全基因组45个SNPs的分型;分型确定C57BL/6, BALB/c, FVB/NJ 等胚胎和精子各10种近交系,SNP位点信息与测序结果一致;小鼠冷冻胚胎个数与SNPs检出个数成正比,当胚胎数达到12以上时SNP检出率100%。结论 实现近交系小鼠冷冻胚胎和精子快速SNP基因分型,及遗传质量鉴定。     

下腹正中小切口兔输精管结扎术

目的探讨下腹正中切口兔输精管结扎术可否减少术后早期并发症。方法正常雄性日本大耳白兔14只(约4月龄),7只(随机选择)经阴囊切口暴露睾丸和输精管后靠近附睾尾进行输精管结扎,另7只从耻骨联合上方的腹正中线上作一小切口,暴露腹股沟管内的输精管后进行输精管结扎。术后10天进行局部解剖,观察手术局部的变化。结果与经阴囊切口相比,经腹正中切口的兔输精管结扎术可明显减轻阴囊水肿以及睾丸、附睾与周围组织的粘连。结论远离阴囊和附睾的经下腹正中切口的兔输精管结扎术,可减轻手术创伤,减少术后早期并发症。     

氩离子激光凝堵输精管后睾丸附睾的形态学观察

对氩离子（Ａｒ＾＋）激光凝堵输精管后不同时期的睾丸和附睾做了形态学观察，发现在术后半月，无论在肉眼和镜下睾丸和附睾均无明显变化，术后１个月，附睾出现淤积现象２～３个月后，睾丸肉眼形态无明显改变，镜下睾丸部分曲细精管生精细胞萎缩，肉眼观附睾体积增大，镜下输出小管和附睾管扩张，个别标志间质内有精子性肉芽肿形成；术后６个月或更长时间，睾丸和附睾的变化基本同前，提示氩离子激光凝堵输精管后，部分睾丸生精细胞     

GFP质粒DNA/阳离子脂质体复合物的制备及其体外表达

目的 为基因免疫和基因治疗寻找一种简单、经济、有效的DNA投递系统。方法 采用逆向蒸发法,用卵磷脂、胆固醇、十八胺制备成阳离子脂质体包封含绿色荧光蛋白基因的真核表达质粒pEGFPC1,对三种不同DNA加入量所形成的DNA／脂质体复合物的微球形、包封率、电荷性、转染真核细胞的效果以及对细胞的毒性作用进行初步检测研究。结果 所获得的DNA／脂质体复合物呈球形、平均粒径为562nm;包封率达55％～65％,为阳离子脂质体;都能有效转染真核细胞,但转染效率有差异,DNA／脂质体混悬液中DNA含量、混悬液的加入量以及细胞的作用时间,都对细胞毒性有不同程度的影响。结论 该方法可作为提高基因免疫效果的简单、经济、有效的手段之一。     

A comparative study on contractile responses of rabbit and guinea pig vasa deferentia to electrical field stimulation

Contractile responses of rabbit and guinea pig vasa deferentia to electrical field stimulation (EFS) are compared. A muscarinic receptor blocking agent, 1 microM atropine markedly reduced phasic and tonic contraction induced by EFS (20 Hz, 0.5 msec, 30 V, for 30 sec) in rabbit vas deferens, while it only slightly depressed those in guinea pig vas deferens. Further addition of an adrenergic alpha1 receptor blocking agent, 1 microM prazosin markedly depressed the second tonic contraction in both rabbit and guinea pig vasa deferentia. In the presence of atropine and prazosin, further addition of a P2X purinoceptor desensitizing agent, 10 microM alpha,beta-methylene ATP (alpha, beta-MeATP) abolished the residual phasic contractile response in guinea pig vas deferens, while it partially depressed that in rabbit vas deferens. The administration of 10 microM alpha,beta-MeATP in the absence of atropine and prazosin markedly potentiated the phasic contractile response of rabbit vas deferens to EFS, while it depressed that of guinea pig vas deferens. Contractile response of rabbit vas deferens to alpha,beta-MeATP was more potent than those of ATP and 2-methyl-thioATP (2-Me-thioATP), while these nucleotides had almost same potency in guinea pig vas deferens. These findings may indicate that contribution of cholinergic, adrenergic and purinergic neurotransmission to the contractile response of rabbit vas deferens to EFS is different from that of guinea pig vas deferens.     

Activation of systemic immune effectors and induction of cytokine production by direct intratumoral injection of IL-2 DNA liposome

ourpreviousstudiesreportedaprotocolwhichreliedonthedirecttransmissionoflL-2geneintoB16F1Otumorsinvivotogeneticallymodifythetumorsastheygrewinsitu.WhenIL-2DNAliposomewasdirectlyinjectedintothetumormassintratumorallyinthemurinemodels,adra-maticinhibitionoftumorgrowthwasobserved-Thesurvivaltimeofthetumor-bearingmicewereprolongedsignificantly.Byanalyzingthefunctionofthetumorcellsand'tumorinfiltratinglympho-cytes(TIL),G418-resistanttumorcellscouldbeseparatedfromthetumormassandhighlevelofIL-2c…     

输精管动脉供应区域的实验研究

目的 探讨同种异体睾丸移植中吻合输精管动脉的临床意义。方法 取去势手术病人睾丸１０只,在１０倍手术显微镜下,经输精管动脉灌注碳素墨水,直接显示输精管动脉的支配区域,结果 输精管动脉支配整个输精管,附睾头、体、尾及部分睾丸实质。结论在同种异体睾丸移植时,为提高睾丸存活和术后生育能力,除进行传统的吻合精索内动脉外,同时行输精管动脉的吻合实属必要。     

Effects of Placental Isoferritin on the Mouse Embryo Development in vitro

To investigate the effect of placental isoferritin （PLF） on mouse embryo development in vitro, mice 2-cell embryos were co-cultured with human first trimester decidual cells at different concentrations of PLF in vitro. The following changes of the above system were observed under an invert microscope and the number of embryos were recorded and the embryos were classified. The results showed there was no significant difference in the percentage of embryos development to 4-cell 8-cell and morula （P〉0.05）. PLF at the doses of 10 and 100 U/mL significantly enhanced more embryos development to the blastocyst and hatching blastocyst （P〈0.05）. PLF at the dose of 1000 U/mL depressed more embryos development from 2-cell to hatching blastocyst, meanwhile such phenomena as cell degeneration and irregular cleavage were observed in part of embryos, but there was no significant difference in statistics （P〉0.05）. It was concluded that PLF at the concentration of 10-- 100 U/mL had no significant effects on the early development of mice embryos, however, PLF could promote the growth, differentiation, and hatching of preimplantion blastocysts.