兔角膜缘干细胞两种培养方法的比较

目的对采用消化法和组织块法培养兔角膜缘干细胞进行对比,以期确定合理的角膜缘干细胞体外培养方法。方法采用3T3成纤维细胞滋养层培养体系,分别用消化法和组织块法培养兔角膜缘上皮细胞,用免疫组织化学技术检测培养的细胞中ΔNp63的表达。采用流式细胞技术分析分离的角膜缘上皮细胞悬液中间质细胞的含量,采用扫描电镜观察去除上皮的角膜缘基质表面。结果采用消化法培养,培养的细胞中ΔNp63表达较多,细胞最终可以分化为复层上皮结构。在分离的细胞悬液中,间质细胞含量小于5%,扫描电镜显示角膜缘上皮细胞能被完全消化。结论在角膜缘干细胞的培养中,在干细胞的含量方面,消化法培养要明显优于组织块法培养。     

同一猴、兔眼角膜细胞成分原代培养的实验研究

目的建立从同一角膜片上分离培养角膜上皮、基质和内皮细胞的方法,为研究角膜细胞间的相互作用机制奠定基础。方法采用消化法分离培养猴眼角膜内皮细胞、上皮细胞和兔眼角膜上皮细胞．组织块培养法培养基质成纤维细胞和兔角膜内皮细胞。细胞接种于12孔培养板,并于培养不同时间行Wright染色检查细胞生长情况。结果采用消化法和组织块培养法能成功地培养猴、兔角膜上皮、基质和内皮细胞。猴、兔角膜内皮细胞培养1wk后,均能形成内皮细胞单层,细胞类似天然的六角形态．且以猴内皮细胞明显;猴角膜上皮细胞培养3～4d生长旺盛、但难以形成细胞单层,兔上皮细胞生长旺盛,1wk后已达融合状态,细胞呈膜状伸出板层伪足;猴、兔基质成纤维细胞易培养,1wk后已融合成单层细胞,细胞排列整齐,类似纤维走行样外观。结论从同一角膜材料中能分别培养出角膜3种细胞成份,消化法和组织块培养法相结合既能节省材料,又简单易行。     

组织块联合胰酶消化法培养兔角膜缘干细胞的初步探索

目的：建立一种简便、高效的兔角膜缘干细胞原代培养方法。

方法：获取兔角膜缘组织,采用组织块联合胰蛋白酶消化法进行兔角膜缘干细胞体外培养,倒置显微镜下观察细胞生长情况,HE染色观察细胞形态和结构特点,并运用免疫组织化学技术进行细胞鉴定。

结果：采用组织块联合胰蛋白酶消化法可以简便、快速地培养出兔角膜缘干细胞,显微镜下动态观察细胞生长良好,有较高的增殖能力; HE染色证实细胞形态和结构正常; AE5及P63免疫组织化学鉴定呈阳性。

结论：采用组织块联合胰蛋白酶消化共同培养的方法,建立了一种简便、高效的兔角膜缘干细胞原代培养模式。     

PHEMA对兔角膜成纤维细胞增殖度的影响

目的 通过材料浸渍液法测定兔角膜成纤维细胞的相对增殖度来评价微孔和无孔聚甲基丙烯酸-β-羟乙酯(PHEMA)的生物相容性。方法 用消化法原代培养兔角膜成纤维细胞，分别将微孔PHEMA和无孔PHEMA浸入细胞培养基24h并用此浸渍液培养兔角膜成纤维细胞，分别于2、4、7d后用分光光度计测定吸光度并计算细胞相对增殖度。结果微孔PHEMA组及无孔PHEMA组的细胞相对增殖度均大于80％，细胞毒性为一级。结论微孔PHEMA和无孔PHEMA对兔角膜成纤维细胞生长影响较小。     

结膜松弛症球结膜成纤维细胞的原代培养及形态学观察

目的:观察原代培养的结膜松弛症(CCH)球结膜成纤维细胞生长状况及形态变化,确定最佳传代时间以获得稳定、一致的CCH球结膜成纤维细胞。方法:采用组织块贴壁法获得CCH原代球结膜成纤维细胞,胰蛋白酶差速消化法进行成纤维细胞纯化,倒置显微镜下观察并记录不同时期成纤维细胞的生长状况及形态变化,免疫荧光细胞化学染色行成纤维细胞鉴定。结果:CCH结膜组织贴壁24h即可见少量细胞从组织块周围爬出,第2～7d为细胞生长对数期,细胞生长快、增殖旺盛,轮廓清晰,分布均匀,数目增多,细胞核清晰;第9～15d细胞生长进入平台期,组织块逐渐老化失去活性,细胞增长缓慢,排列疏松,体积变大,形状扁平,细胞浆内见大量颗粒状物质和小泡产生,部分细胞从培养瓶底脱落,细胞之间出现较大空隙。传代纯化后细胞的大小、形态基本一致,经鉴定为成纤维细胞,呈长梭形、扁平星状或多突的纺锤形,中间宽大,有卵圆形细胞核,两头相对细小,伴向外伸出2～3个长短不一的细长突起。结论:采用组织块贴壁法可成功获得原代CCH球结膜成纤维细胞,当细胞生长至第8d时行消化、传代可获得稳定、一致的CCH球结膜成纤维细胞。     

人翼状胬肉细胞的分离和培养

目的:探索人翼状胬肉细胞的体外分离和培养方法。方法:取原发翼状胬肉50例,无菌条件下将胬肉组织剪成1×11／mm2的组织块,37℃环境中 0．025％Ⅱ型胶原酶消化20 min,0．05％胰蛋白酶消化10 min,1500 rpm离心10 min,将收集到的细胞用内皮细胞条件培养基制成悬液后进行接种培养,经机械刮除和密度梯度离心法进行纯化,采用CD31、CD34、Ⅷ因子免疫组化和透射电镜方法进行鉴定,设阴性和空白对照。结果:经原代培养后可得到人翼状胬肉成纤维细胞和内皮细胞样细胞,胞浆内分别含有VIM 和CD31、CD34及W-P小体。结论:本实验培养获得了人翼状胬肉成纤维细胞和内皮细胞样细胞,方法简便,可操作性强, 为进一步研究翼状胬肉的发病机制奠定了坚实的基础。     

依地酸二钠在兔结膜成纤维细胞体外培养中的应用

目的：研究EDTA溶液在体外培养兔结膜成纤维细胞的应用。方法：将无菌条件下取得的兔结膜用2.5g/L的胰蛋白酶消化后，置于50mL/L CO2、37℃培养箱中，在细胞贴壁长满后以0.4g/L EDTA溶液消化，传代、接种，共18瓶。兔的另1眼结膜用2.5g/L的胰蛋白酶作传代消化液，同样条件培养18瓶。接种48h后观察2组结果。结果：EDTA组兔结膜成纤维细胞生长良好，胰蛋白酶组18瓶中有6瓶生长不良。结论：0.4g/L EDTA溶液用于兔结膜成纤维细胞培养传代时的消化，可减少消化时对细胞的损伤。     

不同成分培养基对原代兔晶状体上皮细胞生长的影响

目的:研究不同培养基对兔晶状体上皮细胞(LEC)培养的影响。方法:使用组织块贴壁法原代培养兔LEC,倒置显微镜观察不同培养基(DMEM低糖、高糖、F12)下兔LEC形态、生长速度的情况。结果:DMEM低糖和高糖培养基使培养2wk后的细胞开始出现分化,并停止生长,晶状体上皮细胞明显成纤维细胞化。DMEM/F12培养基使细胞生长良好,传至第5代时细胞开始发生转化变为成纤维细胞。结论:DMEM低糖和高糖培养基造成兔LEC增殖抑制,DMEM/F12培养基适合兔LEC的生长。     

兔眼Tenon囊成纤维细胞体外培养方法的研究

探索体外培养兔眼Ｔｅｎｏｎ囊成纤维细胞的简便方法。把兔结膜和结膜下组织剪碎后直接接种在培养瓶中,加入ＤＭＥＭ培养液进行培养,省略胰蛋白酶消化过程或者组织块干固贴壁过程。此方法简单而且有效。本文对此进行了详细的介绍,同时还介绍了如何分离培养时混杂生长的结膜上皮细胞,并且对体外培养的兔Ｔｅｎｏｎ囊成纤维细胞的生长速度进行了研究。     

人眼Tenon's囊成纤维细胞原代培养的研究

目的探讨体外培养原代人眼Tenon’s囊成纤维细胞的方法及其生长特性,为抗纤维化研究提供靶细胞模型。方法取翼状胬肉及斜视手术患者的Tenon’s囊组织,用组织块法培养原代成纤维细胞。用免疫荧光方法鉴定细胞。对细胞进行传代、冻存和复苏的观察。对复苏后的细胞行MTT法检测其活力并绘制生长曲线。结果人眼Tenon’s囊成纤维细胞可以在体外用组织块方法培养出来,呈典型的长梭形。免疫荧光鉴定细胞波形蛋白染色阳性。细胞多次传代后依然生长迅速,3 d即可长满瓶底。复苏后细胞2~6 d处生长对数期,增殖能力良好。结论人眼Tenon’s囊成纤维细胞体外生长状态良好,可以液氮冻存,复苏后细胞活力较强,可以用于抗纤维化增生的基础研究。     

Strabisme Alternant - Etude clinique - traitement

The author defines motor and sensory alternation: the term alternation should not be used in isolation, it should always be accompanied by the name of the parameter concerned. Sensory alternation is always found together with motor alternation but the reverse is not true.The examining criteria for a diagnosis of sensory alternation are given, sensory alternation must not be confused with alternating inhibition. Working from clinical observations of cases of motor alternating strabismus, the author selects 2 types of binocular sensory relations which allow one to differentiate between:- cases of primary alternating strabismus- cases of secondary alternating strabismusThese forms will develop in different ways; in both cases a cure is possible providing that the right treatment is prescribed and once prescribed carefully followed, etc. It is always a case of serious forms of strabismus whose developmental period is spread over several years.According to the authors, the frequency of cases of true primary strabismus is from 1–3%, the frequency of cases of secondary alternating strabismus varies according to the type of therapy practised on cases of monocular strabismus with amblyopia. These latter will become cases of alternating strabismus under the influence of certain types of therapy carried out over several years (penalization, rocking, alternated occlusion, etc...).Experimental data on kittens confirm clinical data; kittens placed in abnormal environments during the sensitive period will show modification in the distribution of cortical cells and the absence of binocular cells (either because the excitation of the two eyes was not simultaneous, or not identical: artificial strabismus, occlusion, opaque glasses). This disturbances become irreversible after a certain period of exposure (a function of age, length of exposure, etc...).It is thus necessary to bear in mind: 1) the iatrogenic risks of certain orthoptic treatments, 2) the necessity for a binocular form of treatment as soon as possible, as once a certain stage is passed, cortical plasticity diminishes and the elaboration of normal binocular relations becomes impossible.

---

     

Effects of Adenosine Agonists on Intraocular Pressure and Aqueous Humor Dynamics in Cynomolgus Monkeys

The effects of single or multiple topical doses of the relatively selective A₁adenosine receptor agonists (R)-phenylisopropyladenosine (R-PIA) and N⁶-cyclohexyladenosine (CHA) on intraocular pressure (IOP), aqueous humor flow (AHF) and outflow facility were investigated in ocular normotensive cynomolgus monkeys. IOP and AHF were determined, under ketamine anesthesia, by Goldmann applanation tonometry and fluorophotometry, respectively. Total outflow facility was determined by anterior chamber perfusion under pentobarbital anesthesia. A single unilateral topical application of R-PIA (20–250 μg) or CHA (20–500 μg) produced ocular hypertension (maximum rise=4.9 or 3.5 mmHg) within 30 min, followed by ocular hypotension (maximum fall=2.1 or 3.6 mmHg) from 2–6 hr. The relatively selective adenosine A₂antagonist 3,7-dimethyl-1-propargylxanthine (DMPX, 320 μg) inhibited the early hypertension, without influencing the hypotension. Neither 100 μg R-PIA nor 500 μg CHA clearly altered AHF. Total outflow facility was increased by 71% 3 hr after 100 μg R-PIA. In conclusion, the early ocular hypertension produced by topical adenosine agonists in cynomolgus monkeys is associated with the activation of adenosine A₂receptors, while the subsequent hypotension appears to be mediated by adenosine A₁receptors and results primarily from increased outflow facility.