Antenatal taurine reduces cerebral cell apoptosis in fetal rats with intrauterine growth restriction

From pregnancy to parturition, Sprague-Dawley rats were daily administered a low protein diet to establish a model of intrauterine growth restriction. From the 12th day of pregnancy, 300 mg/kg taurine was daily added to food until spontaneous delivery occurred. Brain tissues from normal neonatal rats at 6 hours after delivery, neonatal rats with intrauterine growth restriction, and neo- natal rats with intrauterine growth restriction undergoing taurine supplement were obtained for fur- ther experiments. The terminal deoxyribonucleotidyl transferase （TdT）-mediated biotin-16-dUTP nick-end labeling assay revealed that the number of apoptotic cells in the brain tissue of neonatal rats with intrauterine growth restriction significantly increased. Taurine supplement in pregnant rats reduced cell apoptosis in brain tissue from neonatal rats with intrauterine growth restriction. Immu- nohistochemical staining revealed that taurine supplement increased glial cell line-derived neuro- trophic factor expression and decreased caspase-3 expression in the cerebral cortex of intrauterine growth-restricted fetal rats. These results indicate that taurine supplement reduces cell apoptosis through the glial cell line-derived neurotrophic factor-caspase-3 signaling pathway, resulting in a protective effect on the intrauterine growth-restricted fetal rat brain.

Dexmedetomidine mitigates isoflurane-induced neurodegeneration in fetal rats during the second trimester of pregnancy

Dexmedetomidine has significant neuroprotective effects. However, whether its protective effects can reduce neurotoxicity caused by isoflurane in fetal brain during the second trimester of pregnancy remains unclear. In this study, timed-pregnancy rats at gestational day 14 spontaneously inhaled 1.5% isoflurane for 4 hours, and were intraperitoneally injected with dexmedetomidine at dosages of 5, 10, 20, and 20 μg/kg 15 minutes before inhalation and after inhalation for 2 hours. Our results demonstrate that 4 hours after inhaling isoflurane, 20 μg/kg dexmedetomidine visibly mitigated isoflurane-induced neuronal apoptosis, reversed downregulation of brain-derived neurotrophic factor expression, and lessened decreased spatial learning and memory ability in adulthood in the fetal rats. Altogether, these findings indicate that dexmedetomidine can reduce neurodegeneration induced by isoflurane in fetal rats during the second trimester of pregnancy. Further, brain-derived neurotrophic factor participates in this process.

Expression of c-Fos protein and nitricoxide synthase in neurons of cerebral cortex from fetal rats in hypoxia and protective role of Angelica sinensis

BACKGROUND： Both c-Fos protein and nitricoxide synthase （NOS） have been used as general indexes in relative research about neurons, but it is lack of reports that c-Fos protein and NOS are applied synchronously to study the neurons of hypoxic fetal rats in uterus. OBJECTIVE： To study the effect of hypoxia in uterus on the expression of c-Fos protein and NOS in neurons of cerebral cortex from fetal rats and whether Angelica sinensis has the protective effect on these neurons in hypoxia. DESIGN： Randomized control experiment.SETTING ： Department of Histology and Embryology, Luzhou Medical College.MATERIALS ： Twelve adult female Wistar rats in oestrum and 1 male Wistar rat with bodymass from 220 to 250 g were chosen. Parenteral solution of Angelica sinensis mainly contained angelica sinensis, 10 mL/ampoule, was provided by Department of Agent of the Second Hospital Affiliated to Hubei Medical University （batch number： 01062310）. METHODS ： This experiment was completed in the Department of Histology and Embryology of Luzhou Medical College from September 2003 to June 2004. ①Twelve adult female Wistar rats in oestrum and 1 male Wistar rat were housed in one rearing cage. Vaginal embolus was performed on conceive female rat at 8： 00 am next day. On the 15^th conceiving day, all conceiving rats were divided randomly into three groups： control group, hypoxia group and Angelica group with 4 in each group. Rats in hypoxia group and Angelica group were modeled with hypotonic hypoxia in uterus. Angelica group： Rats were injected with 8 mL/kg Angelica sinensis injection through caudal veins before hypoxia. Hypoxia group： Rats were injected with the same volume of saline. Control group： Rats were not modeled and fed with normal way. ② Twenty embryos of rats were chosen randomly from each group and then routinely embedded in paraffin. Paraffin sections were cut from the brain of embryos to anterior fontanelle. Double-label staining was used to detect the expression of nNOS and c-Fos in neurons of cerebral cortex from embryos of rats. OLYMPUS Bx-50 microscope was used to observe sections and DP12 digit camera was also used under 400 times to detect types of cells. Under microscope, the number of c-Fos, NOS, c-Fos/NOS positive neurons in cerebral cortex from embryos of rats were counted in 2 fields with magnification of 400 in one section per animal. ③ The data in experiments were analyzed by one-way analysis of variance （ANOVA） followed by q test. MAIN OUTCOME MEASURES： ① Results of immunohistochemical double-label staining of c-Fos/NOS from cerebral cortex; ② Comparison of amount immunohistochemical double-label staining of c-Fos/NOS positive cells from cerebral cortex. RESULTS：① The positive NOS cells and c-Fos/NOS cells in the three groups were mainly distributed in cerebral cortex, but positive c-Fos neurons were not observed. ② Positive NOS cells and c-Fos/NOS cells in hypoxia group were more than those in control group （76.55±12.02, 50.45±10.39; 33.35±7.42, 26.35±6.67, P 〈 0.05）, but those in Angelica group were less than those in hypoxia group （51.70±9.82, 35.65±8.37, P 〈 0.05）. CONCLUSION： Hypoxia can stimulate the increase of expression of c-Fos protein and NOS in neurons of cerebral cortex. However, Angelica sinensis can decrease this expression so as to play a protective role in cerebral neurons of hypoxic fetal rats.

EXPERIMENTAL STUDY IN DIFFERENT EXPRESSION AND ORGANIZATION OF COLLAGEN IN SKIN WOUNDS OF ADULT AND FETAL RATS

Objective To observe the spatial and temporal distribution of collagen in fetal and adult rats wounds. Methods The organization of collagen deposition in fetal and adult rats skin wounds were observed by using van Gieson stain. The methods of immunohistochemistry and in situ hybridization were applied to examine collagen type Ⅰ and Ⅲ peptide and mRNA localization at serial time point during wound healing. Results Collagen type Ⅰ and Ⅲ were present in wounds of both fetal and adult rats, but the timing and pattern of collagen deposition varied. In the fetus, collagen wes detected by 48h postwounding (PW), but uns not present in the adult wounds until 5d PW. N in situ hybridization, signals in the area of the fetal wound were clearly greater and with increased number of cells as compered to that in the adjacent unwounded tissue. Adult rat wounds had evidence in increased signals of procollagen type Ⅰ and Ⅲ production by wound fibroblasts on day 5. Collagen deposited and wes arranged in reticular pattern as that of the nounal in fetal wounds. While in the adult wound, collagen deposited in the fashion of course bundles. bundles Conclusion Fetal rat wounds appeared to produce collagen mainly by an increased number offibroblasts in the area of the wound. In contrast, adult rat wounds underwent fibroblast migration and induction of procollagen mRNA synthesis. Our results Suggest that the deposition of collagen type Ⅰ and Ⅲ is regulated by their gene expression. Chllagen type Ⅲ plays an important role in the arrangement of collagen depoition.

An improved primary culture method for hippocampal neurons in fetal rats and MAP2 identification

目的：建立一种简单、高效、高纯度的胎鼠海马神经元的原代培养方法.方法：取胎龄18天的Wistar大鼠,显微镜下分离脑组织,采用Brain Dissociation Kit消化获得单个细胞;分别观察培养后24h、3d、5d海马神经元的基本形态结构;培养7d后采用微管蛋白相关标志物2（MAP2）鉴定培养神经元的纯度.结果：此培养方法获得的海马神经元状态良好,生长旺盛.培养第7天,经鉴定,神经元的纯度高达99.62%以上.结论：该方法操作简单、高效,所得神经元纯度高,结果稳定.

Cigarette Smoke Induces Apoptosis by Activation of Caspase-3 in Isolated Fetal Rat Lung Type II Alveolar Ep-ithelial Cells <i>in Vitro</i>

Smoking during pregnancy is a major source of fetal exposure to numerous harmful agents present in tobacco smoke. Lung development involves complex biochemical processes resulting in dramatic changes which continue even after birth. In addition to type I cells which form the blood-air barrier, type II alveolar epithelial (AE) cells have important and diverse functions related to immunological protection and stabilization of the alveolus through synthesis and secretion of the pulmonary surfactant. Apoptosis or programmed cells death is an important physiological process during lung embryogenesis and for the proper maintenance of homeostasis. Caspases are proteases that play important roles in regulating apoptosis. Caspase-3 is the key executioner caspase in the cascade of events leading to cell death by apoptosis. We explored the hypothesis that cigarette smoke extract (CSE) induces apoptosis in fetal rat lung type II AE cells by activation of caspase-3. To analyze these factors, isolated fetal rat lung type II AE cells were used. The cells were exposed to different concentrations of CSE (5%, 10% or 15%) (v/v) for 60 min. The results of the present study showed that CSE induced apoptosis in fetal rat lung type II AE cells with a significant increase (p 0.05) in caspase-3 activity and decrease in cell proliferation at CSE concentrations of 10% and 15% (v/v). These observations indicate that cigarette smoke extract induces apoptosis by activation of caspase-3 in fetal rat lung type II AE cells in a dose-dependent manner and may potentially alter the regulated development of the lung and the appearance of the surfactant-producing type II alveolar cells which are critical for the establishment of adequate gas exchange at birth.

EFFECTS OF DAURISOLINE ON CYTOSOLIC FREE CALCIUMIN FETAL RAT CEREBRAL CELLS

Cytosolic free Ca([Ca]i)was measured in dissociated cerebral cells isolated from fetal rats with the fluorescent indicater fura-2. Increase in[Ca]i occurred rapidly following explsure of the cells to 50 mmol/L KCI,10mol/L Bayk 8644 or 200μmol/L glutamate（Glu）.[Ca]i elevated by

Effects of MK-801 on apoptosis of spinal cord neurons after cord injury and fetal cord transplantation in rats

Objective: To study the effects of MK-801, an antagonist to N-methyl-D-aspartate (NMDA) receptors, on the apoptosis of spinal cord neurons after cord injury and fend cord transplantation in rats. Methods: Wistar rats were random- lzed into group A in which the animals were inflicted with spinal cord hemisection and treated with fetal cord transplantation and MK-801, group B in which the fats were injured with cord hemisection and beated with fend cord transplantation but no MK-80l are given and group C in which the rats received similar cord injury and the eavity in their cord was filled with gelfoam. All the rats were .killed on the lst, 3rd, 7th and 14th day after surgery respectively. The sections of the injured segment of the spinal cord were studied with TUNEL (terminal deoxynucleotidal transferase-mediated DUTP-biotin nick end labeling) and the expression of Bcl-2 was observed with immunohistochemistry. The positive cells were quantitatively analyzed with a computer image analysis system. Results: The Seventy of apoptosis of the cord neurons was in the order of group C > group B > group A (P < 0.005) while the ode of the intensity of Bcl-2 expression was grouP A > group B > group C (P < 0.05). Conclusion: Our findings indicate that fetal cord transplantation and the administration of MK-80l, an antagonist to NMDA receptors can attenuate apoptesis of the cord neurons ther spinal cold injury.

葛根素对妊娠期糖尿病大鼠母体和胎儿的影响

目的观察口服葛根素(puerarin,Pue)对妊娠期糖尿病(gestational diabetic mellitus,GDM)大鼠母体的药效及对其胎儿生长发育的影响,为Pue用于治疗GDM提供参考。方法给已孕母鼠尾静脉注射链脲佐菌素建立GDM大鼠模型,灌胃Pue治疗12 d,记录孕鼠体质量及流产情况,检测孕鼠给药治疗前后的空腹血糖,并分别于给药后的第5天和第10天检测母鼠糖耐量;母鼠怀孕第20天行剖腹产,检测胎鼠血糖含量,并观察胎鼠生长发育情况。测定胎鼠体质量、体长、尾长及胎盘和重要脏器的重量,计算脏器指数。结果与模型组相比,Pue可显著降低GDM孕鼠及胎鼠的空腹血糖,改善孕鼠糖耐量,有效缓解GDM导致的孕鼠体质量过度增加和胎鼠体质量过大的状况,降低流产率;并可逆转GDM引起的胎鼠脑、心、肝等脏器指数下降和肾脏脏器指数升高。结论口服Pue可缓解GDM母体及胎儿的高血糖状态,降低流产率,减少巨大儿的发生,促进胎儿重要脏器的发育。

肿瘤坏死因子-α、可溶性血管内皮生长因子受体-1在妊娠期高血压疾病大鼠血清及胎盘中的表达水平及意义

目的:探讨肿瘤坏死因子-α(TNF-α)、可溶性血管内皮生长因子受体-1(sFlt-1)在妊娠期高血压疾病(HDCP)大鼠血清及胎盘中的表达水平及意义。方法:选择20只孕鼠,随机分为HDCP组和对照组,每组各10只,HDCP组于妊娠14 d时,皮下注射L-精氨酸甲酯,连续7 d,建立HDCP大鼠模型,对照组皮下注射等体积0.9%氯化钠溶液。妊娠21 d时,比较各组大鼠血压、24 h尿蛋白含量,记录胎盘重量、胎鼠重量、胎鼠身长,苏木精伊红(HE)染色观察各组胎盘组织结构,原位末端标记技术(Tunel)检测胎盘组织细胞凋亡情况,蛋白质免疫印迹(WB)法检测胎盘组织TNF-α、sFlt-1蛋白表达水平。结果:HDCP组胎鼠重量、胎盘重量、胎鼠身长低于对照组(均P<0.05)。HDCP组大鼠妊娠21 d血压及尿蛋白含量高于妊娠14 d,HDCP组大鼠妊娠14、21 d血糖及尿蛋白含量高于对照组(均P<0.05)。HDCP组大鼠血清TNF-α、sFlt-1水平高于对照组(均P<0.05)。对照组大鼠胎盘组织形态和结构正常,细胞完整。HDCP组大鼠胎盘组织细胞结构不完整,绒毛数目减少,且绒毛大部分不成熟。对照组胎盘组织凋亡细胞较少,HDCP组胎盘组织细胞凋亡率高于对照组(均P<0.05)。HDCP组大鼠胎盘组织中TNF-α、sFlt-1蛋白表达高于对照组(均P<0.05)。结论:TNF-α、sFlt-1高表达可能与妊娠期高血压疾病发生密切相关。

diminazene aceturate 的增加的剂量: 在女 Wistar 老鼠的不利繁殖效果

【正】Objective:To investigate the effects of comparatively high doses of diminazene aceturate on the reproductive performance of female rats in the early stage of pregnancy.Methods:After oestrus synchronisation and successful mating,20 pregnant female rats were randomly divided into four groups(A-D).Group A rats served as the control and were given single intraperitoneal injection of 0.5 mL sterile water(vehicle only) while groups B.C and D rats were given single intraperitoneal doses of 7,14 and 21 mg/kg body weight diminazene aceturate respectively,on day 7 of pregnancy.The gestation length,litter size and weight at birth,and areas of foetal resorption in the uterus were determined post partum.The post-implantation survival index(%) and the gestation index(group%) were also evaluated for rats in all the groups.Results:There was a graded increase in the number of observed resorbed foetuses as the dose of diminazene aceturate was increased,although only groups C(14 mg/kg) and D(21 mg/kg) revealed a significant decrease(P【0.01,ANOVA) in the post implantation survival index of rat embryos.There was also a significant decrease(P【0.05) in the litter weights of rats in groups C and D.Conclusions: Although the pregnant rats showed no overt signs of systemic toxicity even at the highest dose of 21 mg/kg body weight diminazene aceturate in this study,it was concluded that the use of high doses of diminazene aceturate in an effort to combat resistant trypanosomes could have adverse reproductive effects on female animals in the early period of pregnancy.

Alteration of mitochondrial function in adult rat offspring of malnourished dams

Under-nutrition as well as over-nutrition during pregnancy has been associated with the development of adult diseases such as diabetes and obesity.Both epigenetic modifications and programming of the mitochondrial function have been recently proposed to explain how altered intrauterine metabolic environment may produce such a phenotype.This review aims to report data reported in several animal models of fetal malnutrition due to maternal low protein or low calorie diet,high fat diet as well as reduction in placental blood flow.We focus our overview on the β cell.We highlight that,notwithstanding early nutritional events,mitochondrial dysfunctions resulting from different alteration by diet or gender are programmed.This may explain the higher propensity to develop obesity and diabetes in later life.

A new animal model for uterine torsion and uterine ischemia-reperfusion studies, but not fetal hypoxia studies

The aim of the present study was to develop a new animal model for use in uterine torsion, uterine ischemia-reperfusion, and fetal hypoxia studies in rats. A total of 14 pregnant rats on their 18 th-19 th gestational days were used. The animals were randomly divided into two groups: those undergoing the shame operation(group 1),and those in which a 360 uterine torsion was performed using a novel technique,which was corrected 6 hours later(group 2). Subsequently, seven female and seven male rat pups aged 1 month were separated from the mothers in each group. The female rats were monitored until puberty via measuring the vaginal apertures. The 1-month old male rats and the female rats on reaching puberty were decapitated and histopathological tests were performed on the dissected organs, including the cerebral, visceral and genital organs. At the end of the study, no differences were observed between the groups with regard to abortions, offspring death rates and congenital abnormalities. It was observed that the time to reach puberty in female rats born from mothers with uterine torsion was longer, but the difference was statistically insignificant. No microscopic lesions were detected in the cerebral, visceral or genital organs of the offspring. Accordingly, it was concluded that offspring of mothers with the uterine torsion were not affected, at least in the short term. It was generally concluded that this animal model is appropriate for use in uterine torsion and ischemia-reperfusion studies, but is not appropriate for fetal hypoxia studies.

Age-related changes in cerebral angiogenesis and fetal liver kinase-1 expression after cerebral ischemia/reperfusion

Cerebral angiogenesis in the early stages after cerebral ischemia injury is essential for the recovery of nerve function,in which fetal liver kinase-1（Flk-1）,as a major regulator of vasculogenesis and angiogenesis,plays a very important role.Microvessel density（MVD）was greater in an aged model group compared with the young sham operated group（P 〈 0.01）.MVD and the sum of the lumen area were decreased in the aged group at 1,3,6 and 12 days following ischemia/reperfusion（I/R）injury compared with the young model group（P 〈 0.05 and P 〈 0.01,respectively）.Flk-1 protein and mRNA expression was greater in the aged model group when compared with the young sham operated group（P 〈 0.01）.Flk-1 protein and mRNA expression was lower in the aged group at 1,3,6 and 12 days after I/R compared with the young model group（P 〈 0.01）.Flk-1 expression in aged rats attenuated rapidly,but was still maintained at relatively higher levels at 12 days following I/R in younger rats.The results suggest that angiogenesis was weakened after cerebral I/R in aged rats,and the mechanism of which might be correlated with attenuated expression of Flk-1 protein and mRNA.

产前地塞米松暴露对雄性子代大鼠生殖功能的影响

目的 探讨孕中、后期地塞米松暴露对雄性子代大鼠生殖功能的影响。方法 SD孕鼠随机分为对照组和地塞米松产前暴露组,每组各30只。地塞米松产前暴露组大鼠在怀孕第14~18天皮下注射给予地塞米松0.4 mg/(kg·d),对照组大鼠同时期每天皮下注射生理盐水0.5mL/只。怀孕第20天时采用剖宫产的方式取雄性胎鼠,此外让另一部分孕鼠自然分娩,待其子代雄性大鼠90 d后断颈处死。取血液分离血清备用;分离右侧睾丸石蜡包埋,其他的睾丸放置于-40℃冰柜中保存备用。检测血清中激素浓度;检测睾丸组织的甾体合成急性调控蛋白(steroidogenic acute regulatory protein,Star),P450侧链裂解酶(P450 side chain cleavage enzyme,Cyp11a1),3β-hsd;进一步检测精子数量。结果 与对照组相比,地塞米松产前暴露组的胎鼠血睾酮浓度降低(P<0.05),胎睾丸间质细胞数量减少(P<0.05),Star和3β-hsd基因表达水平降低(P<0.05)。地塞米松产前暴露组的成年大鼠血睾酮浓度降低(P<0.05),睾丸间质细胞数量减少(P <0.05),甾体激素合成酶Star和3β-hsd的表达降低(P<0.05),附睾的精子数量降低(P <0.05)。结论 地塞米松产前暴露所致的子代胎儿血清中的睾酮浓度降低并可延续到出生后成年,同时出现精子数量减少,睾丸间质细胞数量的减少以及Star的基因水平表达下降可能是主要机制之一。

胎鼠神经干细胞原代培养及传代条件优化

目的:探讨胎鼠神经干细胞(NSCs)原代培养方法和活性评价体系,确定NSCs的最佳传代条件。方法:选取孕11~14 d SD大鼠,提取胎鼠原代NSCs,采用巢蛋白免疫荧光染色鉴定NSCs。将NSCs以1.0×10^(4)、2.0×10^(4)、6.0×10^(4)、1.0×10^(5)、1.6×10^(5)和2.0×10^(5) mL^(-1)的细胞密度进行传代培养48 h后观察神经球的形态表现,测定神经球直径。活死细胞染色法检测不同直径神经球中NSCs存活率。结果:NSCs呈巢蛋白阳性,培养的细胞为神经干细胞。NSCs呈聚集生长和牢固细胞间黏附聚集模式,形成神经球,平均直径为(152.72±47.52)μm,球与球之间少有单独的细胞散在。与2.0×10^(4) mL^(-1)传代密度比较,6.0×10^(4) mL^(-1)和1.0×10^(5) mL^(-1)传代密度的神经球直径增大(P<0.05)。1.0×10^(5) mL^(-1)、1.6×10^(5) mL^(-1)和2.0×10^(5) mL^(-1)传代密度的神经球直径比较差异无统计学意义(P>0.05)。与0~40μm、40~60μm、60~80μm和80~100μm直径神经球比较,100~200μm直径神经球中NSCs存活率降低(P<0.05)。结论:以1.0×10^(5) mL^(-1)的密度进行传代时神经球直径为80~100μm,可有效提高NSCs传代培养效率和活性。

高原低氧下妊娠大鼠胎盘葡萄糖转运体1的表达及其对胎鼠生长发育的影响

目的:分析高原低氧下妊娠大鼠胎盘组织中葡萄糖转运体1(GLUT1)的表达及其对胎鼠发育的影响。方法:将20只妊娠大鼠随机分为常氧组和低氧组,妊娠第9天常氧组常规饲养(平均海拔2200 m),低氧组饲养于低压氧舱(模拟海拔5000 m),2组妊娠大鼠饲养至妊娠21 d尾静脉采血测定空腹血糖,留取腹主动脉血ELISA法测量血清胰岛素浓度,取出胎盘和胚胎,比较常氧组和低氧组胎盘质量、胎鼠体质量及身长,H-E染色光镜下观察2组胎盘结构,分别采用RT-qPCR和免疫组织化学检测妊娠大鼠胎盘组织中GLUT1 mRNA和蛋白的表达情况,分析胎鼠体质量及身长与妊娠大鼠空腹血糖、胎盘GLUT1蛋白相对表达量的相关性。结果:与常氧组相比,低氧组妊娠大鼠空腹血糖下降,血清胰岛素浓度升高;低氧组胎盘质量无变化,低氧组胎鼠体质量及身长降低。光镜下观察,低氧组滋养细胞排列紊乱,细胞间隙增宽,毛细血管扩张,可见大量红细胞。低氧组妊娠大鼠胎盘组织中GLUT1 mRNA的表达上调;GLUT1阳性细胞分布于滋养细胞、红细胞,主要位于细胞膜,且低氧组GLUT1蛋白表达水平增高。Pearson相关性分析显示胎鼠体质量与妊娠大鼠空腹血糖呈正相关,胎鼠身长与胎盘GLUT1蛋白相对表达量呈负相关。结论:在高原低氧下,妊娠大鼠胎盘组织中GLUT1的表达上调,并且影响胎鼠的生长发育。

鹿茸多肽对胎大鼠脑神经干细胞体外诱导分化的实验研究

目的 探讨鹿茸多肽对胎大鼠脑神经干细胞体外分化的影响。 方法 从E12～ 14d的Wistar大鼠脑中分离扩增获得大量神经干细胞后 ,加入不同浓度的鹿茸多肽 ,观察其对胚胎神经干细胞分化的影响 ,并通过免疫组织化学染色检测神经干细胞分化为神经元和星形胶质细胞的状况。 结果 5 0 μg L组分化细胞总数与对照组相比有显著性差异 (P <0 0 1) ;5 0 μg L、10 0 μg L、2 0 0 μg L组神经元特异烯醇化酶 (NSE)阳性率与对照组相比有显著性差异 (P <0 0 1) ,并呈一定的剂量依赖性。 结论 鹿茸多肽在体外可明显促进神经干细胞向神经元分化 ,为鹿茸多肽应用于神经系统损伤性疾病的治疗提供了实验依据。

胎鼠肺细胞的分离纯化及原代培养

目的 建立一套可靠的胎鼠肺细胞分离、纯化和培养技术 ,用于体外研究肺发育和早产儿肺部疾病。方法采用胰酶和胶原酶消化 19d胎鼠肺组织块 ,经差速离心和反复贴壁法 ,分离、纯化胎鼠肺泡Ⅱ型细胞 (AECⅡ )和肺成纤维细胞 (LF) ,并进行原代培养。用细胞角蛋白 (cytokeratin)和波形蛋白 (vimentin)免疫细胞化学染色和透射电镜对所分离的细胞进行鉴定。结果 该方法所得细胞产量大、纯度高。免疫细胞化学鉴定 ,AECⅡ细胞cytokeratin染色阳性 ,vimentin染色阴性 ,LF则相反。透射电镜观察AECⅡ ,可见细胞内板层小体。结论 该方法是一种可靠的胎鼠肺细胞的分离纯化培养技术 。

改良的胎鼠海马神经元原代培养及模拟糖尿病并发抑郁环境对其的损伤作用

目的:建立一种改良的胎鼠海马神经元原代培养方法,研究模拟糖尿病并发抑郁(diabetes mellitus with depression,DD)环境对其的损伤作用。方法:取胎龄18 d的SD大鼠,体视显微镜下分离海马,0.25%胰蛋白酶和0.2%胶原酶(1∶1)消化后进行体外培养,分别观察培养后4 h、24 h、5 d、8 d、14 d海马神经元的基本形态结构;神经元特异性烯醇化酶(neuron specific enolase,NSE)免疫细胞化学染色进行细胞鉴定。采用高糖联合皮质酮造模构建类似糖尿病并发抑郁症状的模拟DD环境,MTT法检测神经元的存活率;流式AnnexinV/PI双染和Hoechst荧光染色检测神经元的凋亡情况。结果:经NSE免疫细胞化学鉴定,培养7 d后的神经元纯度达95%以上,细胞胞体成球形,树突分支明显,并相互交织成丰富的神经网络;高糖、皮质酮及两者联用造模18 h后,与正常对照组相比,MTT检测细胞存活率分别为53.1%、64.3%和56.7%,P<0.05;流式细胞仪检测正常对照组、高糖组、皮质酮组、两者联用组Q3区的比例(分别为71.6%、47.9%、53.6%和39.2%),明显低于阴性对照组(98.5%),P<0.05。后四者Q2+Q4区总比例分别为24.4%、46.6%、40.4%、67.0%;Hoechst染色结果提示上述三个造模组的神经元细胞核均出现不同程度的核染色质聚集、浓缩,甚至核碎裂,呈现出典型的凋亡特征。结论:成功建立一种操作简便、细胞纯度佳、活性好、产率高的胎鼠海马神经元原代培养方法,验证了模拟DD环境对海马神经元的损伤作用,为体外深入研究DD的发病机制及药物防治提供了实验依据。