染色质

Using GSA-〓 lectin histochemistry, the first appearance of microglia occurred at E13, usually stained as scattered amoeboid cells. From then on, the number of microglia increased steadily, reached a peak at postnatal day 7 (P7), and then reduced gradually to adult level. Generally the morphological form of microglia undergoes transformation from early rounded or ameboid to adult resting ramified as they differentiate during development.

使用凝集素GSA-〓亲和组织化学染色，本实验最早观察到小胶质细胞在胚胎13天（E13），为散在分布的圆形或阿米巴样细胞，随后细胞数量逐渐增多；总体而言小胶质细胞的形态遵循着由圆形或阿米巴样逐渐转向纤细分枝状的演变规律，不同阶段细胞形态各有其特点，在不同区域也存在差异；小胶质细胞的数量在生后第七天（P7）达到高峰，其后开始逐步降低直至成年。

Methods A lamellar flap was produced in the rabbit cornea with a microkeratome. The flap interface in whole corneas and corneal beds were soaked in the rAAV –EGFP solution for 10 min, BSS solution was applied in the control group. The corneas were harvested at day 1, 3, 14, and 21 after application for the structure analysis of cornea by HE staining. Expression of the transferred gene was monitored by fluorescence stereoscope and laser confocal microscope. Results After 3d of infection with rAAV-EGFP ,slight fluorescence was detected in rabbit cornea by fluorescence stereoscope .

微型角膜刀制作兔角膜基质瓣；转染组用含rAAV－EGFP转染液浸泡角膜瓣和角膜基质床10 min，对照组用等量BSS液浸泡角膜瓣和角膜基质床10 min，不同时间点(1、3、7、14、21d)通过荧光体视镜观察角膜出现EGFP荧光的起始时间及分布情况，收集角膜，其中一半用激光共聚焦显微镜观察、照相，另一半固定后行HE染色。

Methods HE and immunohistochemistry were used to observe the changes of glial fibrillary acidic protein and CD34 in the surrounding areas after cerebral infarction in human.

方法采用HE染色观察星形胶质细胞和微血管的变化，免疫组化技术检测胶质纤维酸性蛋白和CD34在星形胶质细胞和微血管的表达。

Following PAS staining, glycogen in hepatoma carcinoma cells mainly expressed near to unilateral cytoplasm surrounding nuclei, showing strong positive expression in the positive control group. Big karyoplasmic ratio and weak expression of glycogen in cytoplasm were visible in the embryonic hepatic progenitor cell group.

PAS染色后阳性对照组肝癌细胞的糖原主要表达在靠近一侧的核周边胞质内，呈强阳性；而胚胎肝前体细胞诱导组，核浆比例大，部分细胞胞质内可见较弱的糖原表达，充满胞质。

At the depigmentation assay, DH and EH show dose-dependently inhibitory activities against mushroom tyrosinase, and the IC50 is 5.697 and 5.256 mM, respectively, and show noncompetitively inhibitory modes in the present of tyrosine, but not DOPA. The control synthetic products, D and E, have no inhibitory effects on tyrosinase. Using tyrosinase activity staining in PAGE gels, both DH and EH show dose-dependently inhibitory pattern in the presence of tyrosine, while, EH also could suppress tyrosinase activity in the presence of DOPA.

而美白活性方面，以商品酪胺酸以商品酪胺酸酵素进行实验，当基质为酪胺酸时，发现DH与EH具浓度相关抑制效果，IC50分别为5.697与5.256 mM，且其相对於基质酪胺酸之抑制型态皆为非竞争型抑制，而其结构类似物之对照化合物D与E则不具此活性；但当基质为多巴时则皆不具此活性；利用原态PAGE电泳活性染色亦发现抑制酪胺酸酵素结果，但EH在基质为多巴时也能下降酪胺酸酵素活性。

After the cell growth curves was recorded, RPE cells of the 3-5th passages were utilized. 2、Three different siRNA (siRNAl,siRNA2,siRNA3) targeting against human cx43 gene and one negative control siRNA were designed and transfected into cultured human RPE cells via liposome reagent. The most effective siRNA can be determined by semi-quantitative reverse transcription PCRRT-PCR. 3、To the most effective siRNA, after transfected into human RPEs with different concentration, the cellular proliferate activities were messured by MTT colorimetry ; the percentages of RPE in different cell circle phase was assayed by FCM; the changes of phenotypical properities were observed with SCM; the protein expression of cx43 was studied through immunocytochemistry stain and Weston blot; the communication intercellular was calculated with FRAP; and the ability of recovery was assessed by using an in vitro wound healing model.4、The total proteins of siRNA1 and RPE were seperated by two-dimensional gel electrophoresis and visualized by silver staining. Proteins with significant expression alterations were selected and their peptide mass fingerprints (PMFs were obtained by matrix-assisted laser desorption/ionization time of flying mass spectrometry (MALDI-TOF-MS).The PMFs were used to search NCBInr database by Auto MS-Fit software.

实验方法：1、培养原代的人RPE细胞，经过细胞角蛋白、S-100和神经胶质原纤维酸性蛋白免疫细胞化学鉴定后，通过AO/PI染色技术确定培养细胞的存活率，描记其生长曲线，第3-5代用于以下细胞实验2、生物合成针对人cx43基因的三条小干扰RNA和一条阴性RNA通过脂质体转染RPE细胞后，通过RT-PCR的方法确定抑制效率最高的干扰片断3、将该片段以不同浓度通过阳离子脂质体转染培养的人RPE细胞后，采用MTT法观察其对细胞的增殖力的作用；通过流式细胞仪观察其对细胞周期的影响；通过扫描电镜观察其对细胞形态的影响；通过免疫细胞化学和Weston blot观察其对cx43蛋白表达的作用；采用激光共聚焦和荧光淬灭恢复技术观察荧光恢复速率平均百分率，评价其对细胞间通讯功能的影响；通过制作RPE细胞损伤模型，观察其对损伤修复能力的作用4、分离纯化转染siRNA的RPE组和正常对照组RPE细胞的全部蛋白质，应用等电聚焦电泳和SDS-PAGE双向电泳技术，银染显示分离出的蛋白质斑点，经凝胶图像分析软件对两个样本进行胶图分析，寻找差异蛋白点。

RESULTS: After differentiation of human adherent myoblasts by neural induction medium, no cells with a neural cell morphology (ie., small, refractile cell body with dendritic cell extensions) were seen. All remaining myoblasts cultured with neural induction medium, myoblasts with proliferation medium and myotubes with differentiation medium containing 20 mL/L HS were positive for β Tubulin Ⅲ. C2C12 myoblasts were negative for β Tubulin Ⅲ. In contrast, all the above cells were negative for the markers Neurofilament Mr 68×103 and GFAP.

结果：用诱导分化液作用后，未见小的、伴有突起的放射状形态的神经细胞；抗β Tubulin Ⅲ对经神经元胶质细胞诱导分化液作用后的人成肌细胞、增殖培养液培养后的各代人成肌细胞及仅含20 mL/L HS分化液分化形成的肌管细胞染色均为阳性； C2C12细胞β Tubulin Ⅲ抗体染色阴性；上述所有细胞抗Neurofilament Mr 68×103和抗GFAP染色均为阴性。

Kangdai Ⅰ has protective function to the damaged neurons and astrocytes: Main results:(1) It has direct protective function to the damaged neurons. It can increase the activity and survival rate, decrease the mortality and the transudation rate of LDH in cultured medium and the strong positive cell count of NOS expression of injured neurons.(2) It has also directly protective function to the damaged astrocytes. It can increase the activity and survival rate and protein content in conditioned medium.(3) It can strengthen the ability of BDNF, GDNF, bFGF, HSP and IL-6expression in damaged astrocytes.(4) It can also strengthen obviously the expressions of NSE, bFGF-receptor and bc1-2, lower the expression of bax and caspase-3.(5) It can indirectly protect and restore the damaged neurons by astrocytes. Because the effect of ACMK (ACM interfered by Kangdai Ⅰ) is stronger than ACM+K (ACM associated with Kangdai Ⅰ).

抗呆Ⅰ号对受损的神经元和星形胶质细胞均具有保护作用：主要表现为：(1)对受损神经元具有直接的保护作用，可提高受损神经元的活性和存活率，降低细胞培养液LDH的漏出率、细胞死亡率和NOS染色强阳性细胞的表达量；(2)对受损的星形胶质细胞也有直接的保护作用，可提高其活性、存活率以及培养液蛋白质的含量；(3)能增强受损星形胶质细胞分泌BDNF、GDNF、bFGF、HSP和IL-6的能力；(4)可明显增强受损神经元对NSE、bFGF的受体和bc1-2的表达，降低受损神经元对bax和caspase-3的表达；(5)抗呆Ⅰ号可通过星形胶质细胞间接地保护和修复受损的神经元，因为在多数实验组中经抗呆Ⅰ号作用的ACM的作用远大于ACM与抗呆Ⅰ号联合应用的作用，统计学上具有显著性差异。

At the third passage, BMSCs showed the typical polar swirl morphology. BMSCs were negative for CD34 and CD45, but positive for CD29 and CD44. Following induction, alkaline phosphatase staining, Oil red staining and toluidine blue staining produced a strong reaction in cells.

第3代骨髓间充质干细胞形态单一均匀，呈典型的极性漩涡状生长，不表达造血前体细胞标志抗原CD34和白细胞标志抗原CD45，表达整合素家族成员CD29和黏附分子CD44，经诱导后碱性磷酸酶染色、油红O染色和甲苯胺蓝染色均呈阳性。

Results 1、 Generally, we can see the original blue and white, shiny, no cracks in the articular surface of the cartilage after the stress increases gradually yellow, surface roughness, cracks appear; when the pressure decreases, the yellowing, rough, the color of the fracture restore gradually and become shiny.2、the shiny smooth surface can be seen under a light microscope, formation, cell distribution, tidy, clear the level of cartilage at the articular surface stress increases, the surface roughness changes, defects, disordered cells, uneven dyeing ; when the articular surface of the pressure gradually decreased, the cartilage gradually repair and the surface of cells at the surface appear only disorder.3、immunohistochemical observation can be seen throughout the observation period, cartilage cells are type Ⅱ collagen expression and expression after 3 weeks gradually weakening, when the seventh week begin to strong gradually.4、 electron microscopy shows that when stress increases the articular surface, the cartilage cells became flat, the cytoplasm in the endoplasmic reticulum, Golgi apparatus decreased with collagen disorders; and when stress decreases the articular surface, cartilage cells gradually returned normal, cytoplasm in the endoplasmic reticulum, Golgi body gradually restore quantity; collagen fibers with a gradual rules.

结果：①大体观察可见到原本蓝白色、有光泽、无裂纹的软骨在关节面压力增大后，逐渐呈灰黄色，表面粗糙，出现裂隙；当压力逐渐减小后，变黄、粗糙、有裂隙的软骨颜色逐渐恢复，变得有光泽②光镜下可见表面光滑、平整，细胞分布均匀、整齐，层次清楚的软骨在关节面压力增大后，表面变粗糙、缺损，细胞排列紊乱、染色不均；当关节面压力逐渐减小后，软骨表面逐渐修复，细胞仅在表层排列紊乱③免疫组织化学观察可见整个观察期内软骨细胞胞浆内均有Ⅱ型胶原表达，术后3周内表达逐渐变弱，从第7周时开始逐渐变强。④电镜下可见当关节面压力增大后，软骨细胞逐渐变扁，胞质中内质网膜、高尔基体减少，胶原排列紊乱；当关节面压力减小，软骨细胞形态逐渐恢复正常，胞质中内质网膜、高尔基体数量逐渐恢复；胶原纤维排列逐渐有规则。

This one mode pays close attention to network credence foundation of the businessman very much.

这一模式非常关注商人的网络信用基础。

Cell morphology of bacterial ghost of Pasteurella multocida was observed by scanning electron microscopy and inactivation ratio was estimated by CFU analysi.

扫描电镜观察多杀性巴氏杆菌细菌幽灵和菌落形成单位评价遗传灭活率。