小鼠腔前卵泡体外发育研究

目的通过小鼠腔前卵泡(preantral     

内分泌和旁分泌因子在腔前卵泡体外培养中的作用

内分泌和旁分泌因子调节着卵泡和卯母细胞发育和成熟。可以对不同时期的卵泡进行体外培养来研究这些因子的作用机制。在卵泡体外培养中.经常使用啮齿类动物作为模型动物,其优势是培养时间短,例如小鼠卵泡的体外生长期为11～12 d,但是人类长达85 d;另外,啮齿类动物卵巢皮质较疏松,卵泡体积较小且数量较少,因此分离小鼠腔前卵泡就相对容易一些;从啮齿类动物试验中所得知的卵母细胞生长因子的作用在人类试验中也得到了证实。本文主要探讨促性腺激素、旁分泌因子、血清和其他添加成分在腔前卵泡生长和分化中的作用。     

获得小鼠初级卵泡进行体外培养的酶解方法比较

目的：对酶解小鼠卵巢,获取初级卵泡的方法进行探讨。方法：卵巢取自性成熟前的昆明系小白鼠,酶液由不同浓度的胶原蛋白酶、脱氧核糖核酸酶和链蛋白酶组成,培养液为Waymouth medium MB752/I,并添加牛血清白蛋白、胰岛素和转铁蛋白。实验共分6组,每组所用酶液组成和处理时间各不相同。结果：由3mg/ml的胶原蛋白酶、100IU／ml的脱氧核糖核酸酶,另加或不加0．02mg/ml的链蛋白酶组成,处理时间为20分钟的两个处理组,卵泡分离效果较好。结论：胶原蛋白酶浓度是影响卵巢中卵泡分离的主要因素,它和消化时间对卵泡分离及分离后卵泡形态、存活能力有影响。     

人成熟卵泡液对不同期未成熟人裸卵体外培养的影响研究

目的探讨人成熟卵泡液对不同时期未成熟人裸卵体外成熟的影响。方法收集卵胞浆内单精子注射(ICSI)治疗周期中未成熟GV期和MI期裸卵,随机分为Ⅰ组(不含成熟卵泡液),Ⅱ组(含成熟卵泡液)组进行体外培养(invitro maturation,IVM),观察不同期裸卵的成熟率,比较不同培养液的体外培养效果。结果 MI期卵与GV期卵相比,24h成熟率(65%比29%)和总成熟率(85%比64.5%),前者均高于后者,有统计学意义(P0.05)。48h成熟率(61.5%比55%),虽然前者高于后者,但差异无统计学意义(P0.05)。两种培养液Ⅱ组与Ⅰ组相比,总成熟率(90.3%比65%)和24h成熟率(64.5%比37.5%),前者均高于后者,有统计学意义(P0.05);48h成熟率(72.7%比44.0%),虽然前者高于后者,但无统计学意义(P0.05)。结论 IVM培养液中添加成熟卵泡液有益于提高未成熟裸卵的体外成熟率,但随着体外培养时间的延长,促进作用可能会减弱。     

睾酮对小鼠卵泡体外发育的影响

目的探讨高浓度睾酮对体外培养的小鼠卵泡生长发育的影响。方法以出生后12日龄的ICR雌鼠体外培养卵泡为研究模型,将卵泡随机分为以下4个组,每组30个:正常对照组(control组)、10-4mol/L睾酮处理组、10-5mol/L睾酮处理组和10-6mol/L睾酮处理组。将卵泡随机分为以下3个组,每组30个:正常对照组(control组)、睾酮处理组(T组,T=10-5mol/L)和雄激素受体拮抗剂氟他胺预处理后再加睾酮处理组(F+T组,F=10-5mol/L,T=10-5mol/L)。在60mm无菌细胞培养皿上进行微滴培养,每天在倒置显微镜下观察卵泡轮廓,颗粒细胞增生情况,卵母细胞形态以及有无卵母细胞逸出等,并记录卵泡存活和退化的情况。结果 10-4mol/L睾酮能抑制卵泡的生长,而10-5mol/L睾酮能够促进颗粒细胞的增殖和卵泡的生长;同时,在卵泡生长前期,睾酮能刺激卵泡的发育,在后期则呈抑制作用,即卵泡发育停滞、退化或死亡。结论在初级和次级卵泡阶段,10-5mol/L睾酮能促进卵泡的发育,而在窦状卵泡阶段则抑制卵泡的发育。     

人表皮细胞体外培养方法的改进

人表皮细胞体外培养方法的改进张慧敏郝振林＊王东霞＊（沈阳医学院附属中心医院医学实验科，烧伤科＊沈阳１１００２４）张志芬王连璞＊＊（沈阳医学院生化教研室，解剖教研室＊＊沈阳１１００３１）表皮细胞体外培养技术的建立和发展对于烧伤治疗、皮肤病学研究等方面，...     

流行性出血热病毒在体外培养的粒—单系前体细胞中增殖的研究

本文用人肺组织和人白细胞制备集落刺激因子(CSF),在体外建立人骨髓粒一单系前体细胞的液体培养。用陈株和C8株两株EHF病毒人工感染,分别在感染后2～4天用免疫荧光法即可检出EHF病毒抗原阳性(EHFV-Ag~+)细胞。随感染时间的延长,Ag~+细胞逐渐增加,荧光亮度增强。将感染病毒的第一代培养物冻融后重新接种新分离的骨髓细胞,第二代感染细胞亦为EHFV-Ag~+,且荧光亮度增强。结果表明EHF病毒能在人粒—单系前体细胞中增殖,并释放感染性EHF病毒。用改良免疫酶染色法观察到骨髓晚幼粒及杆状核细胞的胞浆内有大量的EHFV-Ag,这提示EHF患者外周血中EHFV-Ag~+中性粒细胞可来源于骨髓中感染的粒系细胞。     

人卵丘颗粒细胞和壁层颗粒细胞体外培养方法比较

目的:建立有效分离、提纯及培养人卵丘颗粒细胞的方法,并与人壁层颗粒细胞的体外培养相比较。方法:收集卵胞浆内单精子显微注射取卵时的卵泡液和直接机械分离卵母细胞所得的卵丘颗粒细胞复合物,直接将卵丘颗粒细胞复合物接种于培养皿中培养。用密度梯度离心法分离卵泡液中的人壁层颗粒细胞。用免疫荧光法检测卵泡刺激素受体(follicle stimulating hormone receptor,FSHR)的表达;用CCK-8法检测细胞生长曲线;用ELISA检测这2种颗粒细胞分泌雌激素的能力。结果:直接法所得人卵丘颗粒细胞培养24 h后可贴壁,体外培养生长状态与人壁层颗粒细胞相似;免疫荧光检测显示两者均表达FSHR;CCK-8实验结果表明,两者体外培养生长曲线相似;ELISA结果显示两者分泌雌激素能力相当。结论:利用机械切割获得人卵子周围卵丘颗粒细胞复合物直接培养的方法操作简单,获得的人卵丘颗粒细胞具有与人壁层颗粒细胞相似的生长状态、生长曲线以及雌激素分泌能力,可作为人颗粒细胞亚群体外培养方法的补充。     

人卵的人工受精和体外培养

本实验使用自己配制的T↓6培养液,加人血清白蛋白或母血清,配制成卵泡冲洗液、受精液和生长液。用克罗米芬和HCG诱发超排卵。手淫获得精液、人工获能,以2～3滴精液与1个成熟卵细胞受精。在37℃的CO_2箱中进行培养。受精后16～17 h吹打、换液,这时受精卵已达到原核期;约30 h可达Ⅱ分裂球期;40～44 h为Ⅳ分裂球期;72 h为桑椹胚。在Ⅳ分裂球期进行子宫内胚胎移植。共移植14次,移植胚卵23个,但妊娠尚未能成功。本文对人工受精和体外培养的重要条件进行了探讨,并对人胚早期的发育做了观察。     

人支气管上皮细胞的体外培养

背景：人支气管上皮细胞培养在呼吸系统疾病研究中应用越来越广泛。 目的：探索美国典型培养物保藏中心人支气管上皮细胞体外培养方法的可行性。 方法：对人支气管上皮细胞培养条件进行反复摸索,最终确定应用含体积分数20%胎牛血清的DMEM/F12培养基进行培养。 结果与结论：实验培养的人支气管上皮细胞扁平,呈多边形,长满后呈“铺路石”样分布,经免疫组化检测发现培养的细胞表达上皮细胞标志物细胞角蛋白。在支气管扩张症患者痰液上清刺激下,细胞表达的核因子κB、肿瘤坏死因子α和白细胞介素8明显增强,证实培养的人支气管上皮细胞获得成功。提示不必拘泥于美国典型培养物保藏中心推荐的培养条件,改进的培养方法简便易行,有应用价值。     

Pilot study of isolated early human follicles cultured in collagen gels for 24 hours.

The human ovarian cortex contains mainly primordial and primary follicles. The ability to mature these follicles in vitro could be of great importance for infertility treatments. Fresh and frozen-thawed ovarian tissue was incubated with collagenase and DNase. Follicles with one layer or an incomplete second layer of granulosa cells were then dissected. The follicles were embedded in collagen gels and cultured with Earle's balanced salt solution, 10% fetal calf serum and 0.5 IU/ml follicle stimulating hormone. Increases in the number of granulosa cell layers and in oocyte size were observed in 40 and 38.7% of the follicles from fresh and frozen-thawed tissue respectively, during a 24 h culture period. All the growing follicles were surrounded by cellular outgrowths. Attempts to culture the follicles longer resulted in deterioration of the follicles and oocyte release. Since our study was purely morphological, further growth parameters, e.g. DNA synthesis, should be examined in the future.     

Effects of varying gonadotrophin dose and timing on antrum formation and ovulation efficiency of mouse follicles in vitro

BACKGROUND: This study tested factors affecting mouse follicle growth in vitro, to determine end-points marking follicle function in vitro. METHODS: Pre-antral follicles (mean 137 microm) from B6CBF1 mice were cultured in a substrate-adherent system for < or = 14 days. FSH (0-1000 mIU/ml) day of HCG (1.5 IU/ml days 9-14) protein supplement [fetal calf serum (FCS) (x2) mouse serum (x2) hypogonadal (hpg) mouse serum or human serum albumin (HSA)] were varied. Follicle survival timing of antrum formation incidence of ovulation within 16,24,40,48 h of HCG oocyte growth were assessed. RESULTS: FSH (100 mIU/ml) produced the best antral development (P < 0.001 versus 10 and 1000 mIU/ml). Antra were observed from day 5. Transient antra formed occasionally in the absence of FSH. By 14 days significant senescence had occurred (P < 0.001) but the proportion of follicles ovulating within 16 h of HCG declined from day 9 onwards indicating this to be a more sensitive marker of follicle responsiveness. Optimal growth occurred in 5% FCS (x2) or hpg mouse serum although fewer follicles ovulated in hpg serum (P < 0.05). No normal growth occurred in normal mouse serum (x2) or HSA. Oocytes grew to full size within 9 days with 100 mIU/ml FSH FCS. CONCLUSIONS: These data provide sensitive end-points for assessing follicle growth in vitro.     

Physiology: Growth rates and antrum formation of mouse ovarian follicles in vitro in response to follicle-stimulating hormone, relaxin, cyclic AMP and hypoxanthine

The culture of individual intact follicles in vitro, from smallpre-antral to pre-ovulatory stages, will improve our abilityto perform controlled experiments studying follicle growth andfemale gamete development. This study was undertaken to characterizefurther the conditions required for physiological follicle growthin vitro. We cultured a total of 398 pre-antral follicles ofimmature mice (aged 26–28 days) with an initial diameterof 140–340 µm for 4 days in vitro, using individualmicro-cultures under paraffin oil. Summarizing the results ofall groups, 50 follicles were damaged (12.6%) and, of thoseremaining intact (n = 348), 60 (17.2%) became atretic, 195 (56.0%)became antral and 26 (7.5%) ovulated. The most advanced folliclesgrew to 400–500 µm diameter. The presence of follicle-stimulatinghormone (FSH) in the medium significantly stimulated folliclegrowth in vitro (P < 0.03), in a manner proportional to theinitial diameter over the range of 140–250 µm initialdiameter, with larger follicles being refractory. FSH also significantlyincreased the proportion of follicles forming antra (P <0.001) and their likelihood of ovulating in vitro (P < 0.01),and reduced the frequency of atresia (P < 0.01). Dibutyryl-cyclicAMP mimicked FSH, significantly stimulating growth of largefollicles (P < 0.05) and antrum formation (P < 0.01).Hypoxanthine also stimulated antrum formation (P < 0.01)but did not significantly affect follicle growth. Porcine relaxinhad no significant effect on mouse follicle growth or antrumformation. The optimal conditions for mouse follicle growthin vitro have not yet been defined, but selection of folliclesof < 250 µm diameter and inclusion of FSH or dibutyryl-cyclicAMP in the culture medium are recommended.     

Effects of nerve growth factor and glucocorticoid on cultured human pheochromocytoma cells

Primary cell cultures of two human pheochromocytomas (PC) that were associated with high serum levels of adrenaline and noradrenaline were developed to study the effects of nerve growth factor (NGF) and dexamethasone on the morphology and function of PC cells in vitro. By phase-contrast microscopy, cultured cells were small and hyperchromatic on the first day of culture; neurite-like processes that extended to other cells developed several days later and were maintained for more than 3 months. NGF (100ng/ml), dexamethasone (10^–5M), or NGF + dexamethasone were added to the culture media 2 weeks after the cultured cells had stabilized. Catecholamine concentrations in the medium were maintained at higher levels after addition of NGF, dexamethasone, or NGF + dexamethasone as compared to control cells. In the presence of NGF, extension of neurite-like processes was clearly accelerated, while high levels of dexamethasone inhibited growth of processes. These in vitro studies showed that the addition of NGF or the removal of dexamethasone induces differentiation of adrenal neurons present in pheochromocytomas, suggesting that adrenocortical steroid hormones influence the morphological control of adrenal medullary cells.     

Smoking-associated mitochondrial DNA mutations in human hair follicles

The mitochondrial DNA (mtDNA) of hair follicles was used for studying the genotoxicity of smoking-mediated carcinogens. We determined the incidences of the 4,977 bp and 7,436 bp mtDNA deletions, tandem duplication in the D-loop region and the proportion of the 4,977 bp deleted mtDNA (dmtDNA) in the total DNA of hair follicles from 213 male non-smokers and 74 male smokers, respectively. Twenty-three patients with lung cancer were also investigated. We found that the current cigarette smokers had a 3.1 times higher average incidence of the 4,977 bp dmtDNA (RR: 3.1, P < 0.001) as compared with non-smokers, and this mtDNA deletion was especially prevalent in the old heavy smokers. For the smokers of the age above 70, the average incidence of the 4,977 bp dmtDNA was 3.7 times higher in the group with a smoking index of 401–800 (RR: 3.7, P < 0.005) and 3.2 times higher in the group with a smoking index greater than 800 (RR: 3.2, P < 0.005). However, there was no statistically significant relationship between the incidence of the 7,436 bp dmtDNA and the smoking index, although there was a mild increase in the percentage of the 7,436 bp dmtDNA with the increase of the consumption of cigarettes. No tandem duplication of mtDNA in the D-loop region was disclosed in either smokers or non-smokers group. The proportions of the 4,977 bp dmtDNA in hair follicles were found to correlate with age, but did not keep increasing with cigarette consumption except in the group of subjects with a smoking index of less than 400. On the other hand, we found that the average proportion of the 4,977 bp dmtDNA in the hair follicles was 1.201 ± 0.371% for the patients with lung cancer who had a smoking index greater than 400, while that was only 0.146% for the age-matched healthy smokers with the same smoking index. In conclusion, the high incidence of the 4,977 bp dmtDNA of hair follicles is not only associated with aging but also correlated with the amount of cigarette smoking. A high proportion of the 4,977 bp dmtDNA in the hair follicles may be considered one of the molecular events that are associated with the occurrence of smoking-associated cancers. Environ. Mol. Mutagen. 30:47–55, 1997 © 1997 Wiley-Liss, Inc.     

The prevalence of human thymic lymphoid follicles is lower in suicides

In an autopsy study we determined the prevalence of thymic lymphoid follicles in 311 accident victims in whom the time interval between accident and death was known. We found that the prevalence decreased abruptly in those surviving 48 h or more (P=0.000008). We then compared the prevalence in 271 accident and 168 suicide victims, all of whom had died less than 48 h after the incident and found that the prevalence was significantly lower in the suicide group (P=0.03). We conclude that this difference may be related to the effect on the thymus of high levels of psychological stress likely to have been experienced by the suicides in the days prior to the act. The use of the term hyperplasia to indicate the presence of lymphoid follicles in the thymus and the methodology appropriate for determining the prevalence of thymic lymphoid follicles are discussed. Received: 9 February 1999 / Accepted: 26 August 1999     

Survival of human pre-antral follicles after cryopreservation of ovarian tissue, follicular isolation and in vitro culture in a calcium alginate matrix

BACKGROUND: Ovarian tissue cryopreservation is a promising technique tosafeguard fertility in cancer patients. However, in some typesof cancer, there is a risk of transmitting malignant cells presentin the cryopreserved tissue. To avoid such a risk, pre-antralfollicles could be isolated from ovarian tissue and grown invitro. On the basis of this assumption, the aim of our studywas to investigate in vitro survival and growth of pre-antralfollicles after cryopreservation of ovarian tissue and follicularisolation, followed by encapsulation in alginate beads. METHODS: Ovarian biopsies from four patients were frozen and thawed.Pre-antral follicles were then isolated and embedded in an alginatematrix before in vitro culture for 7 days. RESULTS: Small pre-antral follicles (42.98 ± 9.06 µm) fromfrozen–thawed tissue can survive and develop after enzymaticisolation and in vitro culture. A total of 159 follicles wereincubated in a three-dimensional system (alginate hydrogel)and, after 7 days, all of them showed an increase in size (finalsize 56.73 ± 13.10 µm). The survival rate of thefollicles was 90% (oocyte and all granulosa cells viable). CONCLUSION: Our preliminary results indicate that alginate hydrogels maybe a suitable system for in vitro culture of isolated humanpre-antral follicles. However, more studies are required toestablish whether follicular morphology and functionality canbe maintained using this matrix.