柞蚕蛹卵巢细胞的离体培养及其病毒感染试验

对柞蚕蛹卵巢细胞进行离体培养,原代培养出现细胞聚集中心,生长良好的卵巢原代培养细胞一般在1—2个月由可以形成细胞单层进入传代培养。培养的细胞多为圆形上皮样细胞,并以哑铃状、棱形及分枝伸展的方式进行增殖。其原代培养20天以上的单层细胞对同源的柞蚕核型多角体病毒很敏感。病毒接种3—4天后,细胞出现明显的致病变现象,5—6天后,大部分细胞核内形典型的多角体,8天以后核内多角体成熟,致使细胞破裂而释放到培养基中。而且感染后的细胞培养物回接蛹体和原代细胞仍具感染力。     

不同温度对柞蚕核型多角体病毒在樗蚕蛹精细胞系中增殖的影响

本试验用柞蚕核型多角体病毒（Antheraea Pemyi Polyhedrosis Virus简称ApNPV）感染樗蚕蛹精巢细胞系,分别置于0℃,4℃,15℃,20℃,26℃,28℃,30℃,37℃下静止培养,观察细胞病理变化,测定细胞的感染百分率和多角体含量。试验结果表明,不同温度对病毒的感染率及其在细胞中复制有明显的影响。26℃～28℃是柞蚕核型多角体病毒感染樗蚕蛹精巢细胞并形成多角体的最适培养温度,感染率最高,多角体含量也最多。培养温度过低或过高,多角体都不能在细胞内复制。     

环境条件与柞蚕发生核型多角体病的关系

对柞蚕的蛹、卵、幼虫进行不同环境条件处理试验,调查柞蚕核型多角体病自然发病率。结果下列情况促使柞蚕发生较多的核型多角体病:暖茧期蚕蛹和孵卵期蚕卵长期接触高温多湿(27℃、90％)、低温多湿(15℃、90％);低温(7—8℃)抑制蚕卵胚子15天以上;春柞蚕卵在自然室温(13℃—19℃)保存30天以上;出蚕期卵层堆积厚度1.5cm以上;幼嫩饲料;“下河”蚁蚕的河滩畦芽育;五龄饷食蚕受到38℃3小时高温冲击。     

用核型多角体病毒防治舞毒蛾的研究

用舞毒蛾病虫尸稀释液于室内感染其一龄幼虫,半致死浓度(LC_(50))为89个多角体/毫升;感二龄幼虫,LC_(50)为560个多角体/毫升。经二年蚕场防虫试验,以含82—320万多角体/毫升虫尸液防治1—3龄幼虫,效果达81—98％。用虫尸稀释液行小区感染一龄柞蚕及野外蚕场喷毒感染一、二龄柞蚕试验,病毒浓度从56—87,000个多角体/毫升,对柞蚕未见致病性。     

柞蚕核型多角体病毒对柞蚕蛹的感染性试验

苜蓿银纹夜蛾核多角体病毒(AcNPV)和家蚕核型多角体病毒(BmNPV)载体表达系统的建立,使数百种外源基因得到高效的表达.该系统的产业化开发如大量培养昆虫细胞,存在成本高、技术难度大等问题.建立柞蚕核型多角体病毒(Antheraea pernyi nuclear polyhedrosis virus ,ApNPV)载体表达系统,是利用柞蚕蛹为宿主进行外源基因表达.我国柞蚕资源丰富,柞蚕的蛹体大,以蛹滞育越冬,能够满足工厂化生产的需求.柞蚕核型多角体病毒有3个不同亚株,本实验研究了ApNPV A株系[简称ApNPV(A)]对不同发育阶段和不同品种的柞蚕蛹的感染性,为利用柞蚕蛹表达外源基因的产业化开发奠定基础.     

柞蚕对核型多角体经口感染的抵抗性研究

取不同素质的柞蚕,于1、2、5龄饷食期,用离心提纯的新鲜核型多角体病毒液以五倍系列稀释5—6六个浓度,分别进行经口定量添毒测定,然后根据各组发病情况,求出半数致死量(LD_(50)),来衡量各类型柞蚕的抗毒力大小。初步认为:(1)河南一化性柞蚕品种,对核型多角体病毒抵抗性差异在十倍左右。(2)同品种不同蛾系间的抗毒力也有一定差异。(3)辉点蚕的抗毒力是二对侧点蚕<一对侧点蚕<正常蚕。(4)对不同发育阶段的柞蚕,5龄起蚕的抗毒力比3龄起蚕增长47倍;5龄不同发育时期的柞蚕抗毒力的增长,与其体重增长有关。(6)不同外界环境,可导致柞蚕形成不同的生理状态,因此对核型多角体的抵抗力有明显差异;不同卵期经过的柞蚕抗毒力是:20天>30天>40天;不同饲料育的1、2龄柞蚕,不论是先食叶后添毒,或者是先添毒后食叶,其抗毒力均为适熟叶育>幼嫩叶育。     

一例柞蚕核型多角体病毒病的诊断

2019年5月,我国辽宁省丹东市某柞蚕养殖户发生柞蚕大量死亡,疑似发生了柞蚕核型多角体病毒病,该病为柞蚕的一种急性传染病,一直以来对我国柞蚕养殖业造成了巨大的经济损失.本实验首先通过病蚕临床症状和病理切片观察进行初步鉴定,然后进行PCR鉴定,检出了柞蚕核型多角体病毒(ApNPV),同时通过细菌培养发现没有细菌感染.因此,该养殖户柞蚕所患疾病确诊为柞蚕核型多角体病毒病.     

核型多角体病毒感染柞蚕幼虫细胞的电子显微镜观察

用电子显微镜观察了NPV对柞蚕幼虫的脂肪、血球、真皮、气管及中肠等组织细胞的侵入、增殖和细胞的变化。 观察到进入血液中的病毒粒子,随着血液的循环首先聚集于侵染细胞的周围,并以它的先端部吸附于细胞膜上,以它的外膜和细胞膜融通,病毒便钻入细胞质中。 被感染的细胞核内,首先出现染色质凝集现象,接着核膨大,并生成“网状构造”,看到了杆状病毒粒子的复制、成熟和多角体堆积的过程。 经口接种NPV的柞蚕幼虫,中肠皮膜细胞同其它易感细胞一样,容易被侵染;病毒在中肠皮膜细胞核内复制的过程,并不晚于其它易感细胞,但多角体的形成却晚于其它组织细胞。     

柞蚕核型多角体病毒ie-2和pe-38基因的克隆与序列分析

采用随机克隆方法,通过对插入pGEM-3Z载体的柞蚕核型多角体病毒基因组的部分片段进行测序和序列分析,获得了柞蚕核型多角体病毒基因组的2个极早期基因ie-2和pe-38的序列及pe-38的5′启动子区,其推定的开放阅读框分别编码295和302个氨基酸,并且内部含有一个保守的锌指结构和亮氨酸拉链。核苷酸和氨基酸同源性比较结果显示:柞蚕核型多角体病毒的ie-2和pe-38基因与黄杉毒蛾多角体病毒的同源性较高,与家蚕核型多角体病毒的同源性都很低;在分子进化方面,这2个基因的保守性不强,出现大片段缺失,是研究分子进化及物种关系比较典型的基因。     

利用柞蚕核型多角体病毒表达载体系统在柞蚕蛹中表达增强型绿色荧光蛋白

为了探讨外源蛋白在柞蚕蛹-柞蚕核型多角体病毒(ApNPV)表达系统的表达水平和稳定性,将增强型绿色荧光蛋白(EGFP)基因克隆到柞蚕核型多角体病毒转移表达载体pApM748BE中,获得重组质粒DNA,与ApNPV DNA共转染樗蚕(Phi-losamia cynthiam)培养细胞Pc-01后,用末端稀释法筛选获得重组病毒ApNPV-Δph/egfp+。将该重组病毒感染柞蚕蛹,采用SDS-PAGE和Western blot方法检测EGFP在柞蚕蛹中的表达,结果显示:在柞蚕蛹感染重组病毒ApNPV-Δph/egfp+后6 d,EG-FP就有明显的表达;感染后12～18 d,蛹体液中的EGFP含量保持较高水平,以EGFP标准样品定量分析EGFP在柞蚕蛹体液中的表达水平高于1 mg/mL;感染后30～39 d仍可以检测到蛹体液中EGFP的表达,而且蛋白稳定。研究结果表明,利用柞蚕核型多角体病毒作表达载体,可在柞蚕蛹中高效稳定地表达EGFP,并且EGFP在雌雄蛹间的表达水平无明显差异。     

细胞培养基MLM-45及培养条件对不同发育时期柞蚕卵巢细胞生长的影响

选择柞蚕5龄幼虫以及前滞育期、滞育期和解除滞育后发育前期、中期、后期的柞蚕蛹分离卵巢细胞,利用新研制的柞蚕细胞培养基MLM-45,在不同温度、pH值及渗透压条件下进行柞蚕卵巢细胞的体外培养,探究最佳培养条件及卵巢细胞取材的最佳发育时期。用台盼蓝染色活细胞计数方法,测定不同条件下用MLM-45培养基培养的不同发育时期柞蚕卵巢细胞的活力,当在27℃、pH 6.3、渗透压325 mOsm/kg的条件下培养30 d时,上述各发育时期柞蚕卵巢细胞的存活率最高,并且5龄幼虫、前滞育期和解除滞育后发育前期的卵巢细胞出现了增殖,其增殖率分别为54%、64%和2%,滞育期及解除滞育后发育中期和后期的卵巢细胞存活率也分别达到99.6%、96.6%和92.4%。在此最佳培养条件下,各发育时期柞蚕卵巢细胞培养2个月后其形态以圆形和椭圆形为主,尤以5龄幼虫和前滞育期蛹卵巢细胞的培养效果最好,细胞增殖7～8倍,是适于以MLM-45培养基培养的最佳发育时期柞蚕卵巢细胞。     

柞蚕蛹卵巢原代细胞培养方法及培养基的选择试验

采用机械分散法和胰酶消化法,分别选取MGM-448、Grace、TC-100等3种培养基进行柞蚕蛹卵巢组织细胞的体外培养。在2个月的原代细胞培养期间,机械分散法获得的培养物在MGM-448培养基中贴壁效果好,细胞健康且生长旺盛;而在Grace和TC-100培养基中,细胞虽能贴壁,但生长较缓慢。胰酶消化法分散细胞的效果好,但在3种培养基中的细胞会出现轻微损伤而影响活力。综上认为,柞蚕蛹卵巢原代细胞的最佳培养方法为机械分散法,最佳培养基为MGM-448。     

Expression Pattern of Vascular Endothelial Growth Factor in Canine Folliculogenesis and its Effect on the Growth and Development of Follicles after Ovarian Organ Culture

In this study, the expressions of VEGF in dog follicles were detected by immunohistochemistry and the effects of VEGF treatment on the primordial to primary follicle transition and on subsequent follicle progression were examined using a dog ovary organ culture system. The frozen‐thawed canine ovarian follicles within slices of ovarian cortical tissue were cultured for 7 and 14 days in presence or absence of VEGF. After culture, the ovaries were fixed, sectioned, stained and counted for morphologic analysis. The results showed that VEGF was expressed in the theca cells of antral follicles and in the granulosa cells nearest the oocyte in preantral follicle but not in granulosa cells of primordial and primary follicles; however, the VEGF protein was expressed in CL. After in vitro culture, VEGF caused a decrease in the number of primordial follicles and concomitant increase in the number of primary follicles that showed growth initiation and reached the secondary and preantral stages of development after 7 and 14 days. Follicular viability was also improved in the presence of VEGF after 7 and 14 days in culture. In conclusion, treatment with VEGF was found to promote the activation of primordial follicle development that could provide an alternative approach to stimulate early follicle development in dogs.     

鲤鱼鳍条细胞的原代培养

通过对鲤鱼鳍条细胞进行原代培养,摸索出最佳的培养条件。采用组织块培养法,取一小块鳍条组织在不同培养基、温度、pH、CO₂等培养条件下进行培养,对其生长状况进行观察。培养48 h可见组织块周围有细胞迁出,并形成生长晕,培养1周可形成单层细胞,主要由上皮样细胞和少量成纤维样细胞组成。本研究初步确立了鲤鱼的鳍条细胞培养条件为:培养基为M199,培养温度为25~27 ℃,pH为7.2~7.6,CO₂浓度为5%,原代培养血清浓度为15%~20%,连续培养12 d后可得到纯化的鳍条细胞。     

鸡贮精腺上皮细胞的分离及原代培养

试验旨在探讨稳定可靠的贮精腺上皮细胞分离及原代培养方法,为研究鸡贮精机理提供细胞模型。以鸡输卵管的子宫阴道交接部组织样为材料,采用酶消化法和组织块培养法分离培养母鸡贮精腺上皮细胞,观察母鸡贮精腺上皮细胞的培养情况,比较不同细胞培养方法获得贮精腺上皮细胞的生长情况。结果表明,用胶原酶或胰酶单独消化母鸡子宫阴道交接部组织,经100目过滤后获得的贮精腺上皮细胞24 h后可贴壁,但48～72 h后细胞死亡;用胶原酶Ⅺ(0.01 g/mL)与胰酶(0.25%)先后消化母鸡子宫阴道交接部组织后再经100目过滤获得的贮精腺上皮细胞贴壁性良好,24～48 h细胞出现明显增殖,72 h后细胞增殖速度减慢,开始死亡;用组织块培养法7 d可获得鸡贮精腺上皮原代细胞,该细胞可传2～3代;用组织块培养法获得的细胞进行免疫组化试验,发现细胞表达贮精腺差异表达基因编码的NXPH1蛋白,该蛋白在培养细胞内的表达符合其分泌蛋白特性,表明组织块培养法所获细胞可用于后续研究。综上,用组织块培养法获得的鸡贮精腺上皮细胞可为研究母鸡贮精腺机制提供细胞模型。     

梅花鹿鹿茸间充质层细胞的体外培养和冷冻保存

为了研究梅花鹿鹿茸间充质层细胞的生物学特性,取梅花鹿鹿茸生长顶端间充质层组织,分离间充质层细胞进行体外培养及冷冻保存。结果表明:在含10%胎牛血清(FBS)的DMEM培养基中,鹿茸间充质层细胞能进行短期体外培养,培养的细胞呈成纤维细胞样,培养7 d可长至汇合。以含5%二甲基亚砜(DMSO)和10%FBS的DMEM为冻存液,经梯度降温后冻存,间充质层细胞复苏后存活率较高。在4℃条件下,间充质层组织在含10%FBS的DMEM中可保存7 d。     

Effect of growth differentiation factor‐9 (GDF‐9) on the progression of buffalo follicles in vitrified–warmed ovarian tissues

To improve the reproductive performance of water buffalo to level can satisfy our needs, the mechanisms controlling ovarian follicular growth and development should be thoroughly investigated. Therefore, in this study, the expressions of growth differentiation factor‐9 (GDF‐9) in buffalo ovaries were examined by immunohistochemistry, and the effects of GDF‐9 treatment on follicle progression were investigated using a buffalo ovary organ culture system. Frozen–thawed buffalo ovarian follicles within slices of ovarian cortical tissue were cultured for 14 days in the presence or absence of GDF‐9. After culture, ovarian slices were fixed, sectioned and stained. The follicles were morphologically analysed and counted. Expression pattern of GDF‐9 was detected in oocytes from primordial follicles onwards, besides, also presented in granulosa cells. Moreover, GDF‐9 was detected in mural granulosa cells and theca cells of pre‐antral follicles. In antral follicles, cumulus cells and theca cells displayed positive expression of GDF‐9. In corpora lutea, GDF‐9 was expressed in both granulosa and theca lutein cells. After in vitro culture, there was no difference in the number of primordial follicles between cultured plus GDF‐9 and cultured control that indicated the GDF‐9 treatment has no effect on the primordial to primary follicle transition. GDF‐9 treatment caused a significant decrease in the number of primary and secondary follicles compared with controls accompanied with a significant increase in pre‐antral and antral follicles. These results suggest that a larger number of primary and secondary follicles were stimulated to progress to later developmental stages when treated with GDF‐9. Vitrification/warming of buffalo ovarian tissue had a little remarkable effect, in contrast to culturing for 14 days, on the expression of GDF‐9. In conclusion, treatment with GDF‐9 was found to promote progression of primary follicle that could provide an alternative approach to stimulate early follicle development and to improve therapies for the most common infertility problem in buffaloes (ovarian inactivity).     

Research on the isolation of mouse Leydig cells using differential digestion with a low concentration of collagenase

The aim of this study was to establish a novel method for isolating and purifying Leydig cells from mice testes. Testes of postpuberal mice were harvested and digested in a low concentration of collagenase NB4 for 15 min 2 times. Cells obtained were cultured in low glucose DMEM with 10% FBS. Immunofluorescence was used to detect the expression of Leydig cell biomarkers including 3β-hydroxysteroid dehydrogenase, cholesterol side-chain cleaving enzyme (CYP11A1) and 17α-hydroxylase/17,20-lyase (CYP17A1). It was found that the purity of the isolated Leydig cells was 69.6 ± 4.16%. After 7 days in primary culture, it increased to 90%. The testosterone synthase spectrum could be detected at the primary culture. In conclusion, the application of a low concentration of collagenase for differential digestion allows isolation of large quantities of viable Leydig cells.     

Early ovine preantral follicles have a potential to grow until antral stage in two‐step culture system in the presence of aqueous extract of Justicia insularis

The objective of this study was to determine whether preantral follicles cultured in vitro for 7 days within ovine ovarian cortical strips could be isolated at the secondary follicles (SF) and grown until antral stage during an additional 6 days period of in vitro culture in the presence of aqueous extract of Justicia insularis. Fresh ovarian fragments from 16 adult sheep were fixed for histological analysis (Control 1) or in vitro cultured individually in α‐MEM⁺ supplemented with 0.3 mg/ml J. insularis (Step 1) for 7 days. Part of the fragments then were fixed for histological analysis (in vitro culture group). Remaining fragments were exposed stepwise to increasing trehalose concentrations before immediate isolation of SF and viability assessment (Control 2) or after 6 days of culture in α‐MEM⁺⁺ supplemented with 0.3 mg/ml J. insularis (Step 2). In Step 1, percentage of follicular activation was 80%. In Step 2, a significant increase (p < 0.05) in follicular diameter and antrum formation within 6 days in vitro culture of isolated follicles was achieved. The total antioxidant capacity from both steps significantly increase (p < 0.05) from day 2 to day 6. Confocal analysis of oocytes showed 57.14% oocytes with homogeneous distribution and 42.86% with peri‐cortical distribution. In conclusion, SF can be successfully isolated from sheep ovarian cortex after 7 days of culture and are capable of surviving and forming an antral cavity if cultured in vitro for an additional 6 days in the presence of 0.3 mg/ml J. insularis.     

驴皮成纤维细胞的体外培养研究

试验旨在比较不同培养方法对驴皮成纤维细胞培养的效果,以便建立其体外高效快速的培养体系。采集6~7岁健康的新疆驴真皮组织,分别用组织块贴壁法和双酶消化法对驴皮成纤维细胞进行原代培养,并检测分析细胞的生长特点、生长速度、细胞周期与支原体污染等指标。结果显示,双酶消化法（0.25%胰酶处理1 h,再用0.5%胶原蛋白酶Ⅰ处理6 h）培养驴皮组织3 d后,成纤维细胞进入了对数增殖期,而组织块贴壁法则需要15 d;细胞周期经流式分析发现,处于G0/G1期与S+G2+M期的细胞均约为50%,其余5.17%细胞处于凋亡期,表明大多细胞处于分裂增殖的活跃期,细胞具有很强的活力;试验中培养的细胞均无支原体污染。综上,应用双酶消化法可快速、高效地建立驴皮成纤维细胞体外培养体系。