细胞

(1) cerebral ischemical reperfusion injury rats'limbs motor function is variable. Acupuncture could promote lims'functional recovery.(2) PCNA masc cells is visible in cerebral ischemical semidarkness region. There is cell regeneration phenomenon. Acupuncture could strengthen injury region's PCNA expression, could profit injury recovery and functional reconstruction.(3) In ischemia semidarkness region for the model group and acupuncture group, PCNA masc cells percentage of 14days group is lower than 7days group. Along with the recovery of injury, cell multiplication is weaken.(4) In cerebral ischemia semidarkness region, there is VEGF masc cells and regeneration phenomenon. Acupuncture could strengthen injury region's VEGF expression, could profit protection after injury and blood vessel regenerate.(5) In ischemia semidarkness region for the model group and acupuncture group, VEGF masc cells percentage of 14days group is lower than 7days group. Along with the recovery of semidarkness region, ischemia and anoxemia state is getting improved, and VEGF is reduce.(6) As there are PCNA and VEGF masc cells in brain injured region, we could conclude that, after brain ischemical reperfusion injury, there are blood vessel regeneration phenomenon. Acupuncture could promote blood vessel regeneration, recovery blood supply sufficiently and quickly, and promote the recovery of brain injury region.(7)The VEGF masc cells percentage of inhibitor group is lower than acupuncture group. It state that the effect of acupuncture promote VEGF is partly depend on the existing of eNOS.

实验结论：(1)脑缺血再灌注损伤后大鼠的肢体运动功能发生改变，针刺可以促进肢体功能恢复；(2)脑缺损伤区可见PCNA阳性细胞，存在细胞再生现象，针刺可以增强损伤区PCNA的表达，有利于损伤的修复和功能重建；(3)针刺组和模型组14d时缺血损伤区PCNA阳性细胞百分比低于7d组，随着损伤逐渐得到修复，细胞增殖现象减弱；(4)脑缺血损伤区可见VEGF阳性细胞，存在内皮型细胞再生现象，针刺可以增强损伤区VEGF的表达，有利于脑损伤后保护和缺血区血管再生；(5)针刺组和模型组14d时缺血损伤区VEGF阳性细胞百分比低于7d组，随着缺血损伤的修复，缺血缺氧状态得到改善，产生的VEGF减少；(6)由于脑损伤区同时出现PCNA阳性细胞和VEGF阳性细胞，前者是增殖细胞的标志，后者是促进血管再生的重要因子，可以推断，脑缺血再灌注损伤后脑内存在血管再生现象，针刺可以促进损伤区的血管再生，更迅速而充分的恢复损伤区的血供，促进脑损伤区的修复；(7)抑制剂针刺组脑损伤区VEGF阳性细胞百分比与针刺组相比有不同程度的降低，说明针刺促进缺血损伤区VEGF表达部分依赖eNOS的存在。

The rate of c-kit+ cells in the heart increased gradually from E10 with the highest level of 12.6±3.2% at E11, which was nearly similar to the fetal liver, except for E12 when the c-kit+ cells was 3.4±1.2% in the heart compared to the fetal liver with 11.6±4.1% c-kit+ cells. The rate of c-kit cells in the suspension cells from the heart at E11 maintained stable after 24h culture based in the stromal cells of the heart and AGM region with the same conceptus age, but the rate of c-kit cells obviously decreased in the condition of the stromal cells from the fetal liver. The c-kit cells of the three conditions were all at low level at 48h without significant difference.

通过流式检测c-kit+细胞我们发现，胚胎期心脏和胎肝c-kit+细胞比率几乎一致，E11时c-kit+细胞明显增多，分别高达(12.6±3.2)%和(9.6±2.8)%，但E12时心脏中c-kit+细胞明显减少，仅为(3.4±1.2)%，此时胎肝中c-kit+细胞为(11.6±4.1)%。E11的悬浮细胞与心脏内皮细胞和AGM区基质共培养24h时，c-kit+细胞数量保持稳定，但在胎肝基质细胞中c-kit+细胞数量明显减少，在共培养48h，三组中的c-kit+细胞均呈较低水平，组间没有显著性差异。

Cell proliferation and viability were assayed 48h after transfection, and MDA-7 demonstrated selective inhibition of tumor cell growth, inhibitory rates of A549, Hela and HepG2 cell lines were 25%, 20% and 19%, respectively, but had no significant effect on human fetal kidney derived 293 cell line. Hela cell was screened by G418 for 2 weeks after pcDNA3-MDA-7 and monolayer colony was counted, its monolayer colony formation was 30% of cells transfected with pcDNA3. 0 vector. CMV-driven MDA-7 adenovirus vector was constructed. 293 showed no significant apoptosis during adenovirus packaging and the unpurified adenovirus titer was about 1×10〓pfu/ml. Cos 7, A549, Hela, HepG2 and Hep3B cell was infected with Ad-GFP at different MOI.

二。黑色素细胞分化相关蛋白-7（MDA-7）的克隆及功能研究：利用RT-PCR方法从5月龄人胚胎脾细胞扩增MDA-7的编码序列，经测序鉴定序列与文献报道一致后，与真核表达载体pcDNA3.0连接，构建pcDNA3-MDA-7表达载体，瞬时转染293、A549、Hela和HepG2细胞后抽提细胞总RNA，RT-PCR结果显示表达载体可介导MDA-7在不同细胞系中有效表达；转染48h后测定细胞增殖和活力，MDA-7可选择性抑制肿瘤细胞的增殖，对A549、Hela和HepG2细胞的抑制率分别为25％、20％和19％，但是对人胚肾来源的293细胞生长无明显影响。pcDNA3-MDA-7载体转染Hela细胞后，以G418筛选2周后计数单层细胞集落形成数，计数仅为转染pcDNA3.0空载体的细胞的30％左右。

DCs acquired by our reformed methods express both CD83 and CD 14 molecules highly, and have a higher density than other domestic reports. The higher TNF in DCs culture medium of HC patient suggests DCs in patient still have antigen presenting ability and by optimization the culture medium would improve its presenting ability and have a potential value in design and application individual vaccine. Although antigens pulsed DCs have a decrescent antigen presenting ability but BCG HSP70 could induce its mature and improve its presenting ability. Suggests BCG HSP70 would be a useful mature inducer. Lymphocytes primed by DCs based HC vaccine have the specific cytotoxicity against HCC lines. The CTL after freezing and anabiotic could prophylaxis and therapy HC xenograft on nude mouse. The results also suggests that CD4〓 lymphocytes play a important role in HC with a good differentiation and would be useful in treatment this kind of HC. After being activated by Peptide LLNQHACAV of hAFP and apoptotic HCCs pulsed DCs respectively, the culture medium of activated lymphocytes both contains a high level Th1 cytokines IL-12 and TNF. Primed lymphocytes appeared a characteristic of NK cells. DCs not only inhibited the growth of human HCC and other cancer cells in vitro but also prevented the growth of HC xenograft on nude mouse in vivo. There are at least three kinds of mechanism playing important role in DC based vaccine ,there are inhibition of DCs, HC specific CTL and cytokines pathway.

诱导出的DC共同表达CD83和CD14分子，CD83分子表达明显高于国内报道；肝癌患者DC培养上清中TNF水平高于健康人，提示肝癌患者DC仍具一定的抗原呈递能力，适当调控可使其行使APC功能，以期在肝癌个体化疫苗中发挥作用；DC负载肝癌可溶性抗原后，抗原呈递能力降低，BCG HSP70可促进DC成熟，增加其抗原呈递能力，预示BCG HSP70有可能成为促进DC活化和成熟的另一重要分子；肝癌DC疫苗在体外诱导肝癌特异性淋巴细胞，活化的淋巴细胞在体外对肝癌细胞的杀伤以特异性CTL为主，同时分泌较高水平Th1型细胞因子IL-12和TNF，并抑制4种肝癌细胞生长；冷冻复苏后的肝癌特异性淋巴细胞可以预防和抑制人肝癌裸鼠皮下移植瘤，提示DC负载肝癌可溶性抗原后诱发的MHC-Ⅱ类限制性CD4〓T细胞有可能在分化程度高的肝癌治疗中发挥作用；用DC和HLA-A2〓DC分别负载凋亡肝癌细胞和hAFP218-226位LLNQHACAV HLA-A2限制性九肽，在体外诱导肝癌特异性淋巴细胞，活化后的CTL细胞分泌较高水平的Th1型细胞因子IL-12和TNF，并具较强杀伤活性，此CTL同时具备NK细胞特征；DC对肿瘤细胞的抑制作用可能是通过吞噬实现的，Fas-L在DC抑制中也起一定作用；DC对人肝癌裸鼠皮下移植瘤的抑制率为97％；在肝癌DC疫苗的作用中，至少联合3种以上机制，即通过DC的直接作用、肝癌特异性CTL和细胞因子途径直接或间接地杀伤和抑制肝癌细胞。

Fig.1 SHEE cultured on coverslide, the living cells were growing in single layer with rich cytoplasm, the nuclei were uniform in size with a nucleolus ph ×400 Fig.2 SHEE had a nucleus with ellipse shape, large nucleolus and the cytoplasm contained mitochondria and tonofibrilEM ×10 000 Fig.3 SHEE was spherical in shape, with pseudopods attached on petri dish and abundant villi on cell surface SEM ×5 000 Fig.4 Same as in Fig.3, cell attached on petri dish, appeared stellate or polygonal in shape, with abundant pseudopods and cytoplasmic processes. Protrusive nuclear region in central part of the cell had more micro-villi SEM ×5 000 Fig.5 Chromosomes of SHEE cells belonged to diploidy type Giemsa ×1 000 Fig.6 The SHEE cells of stained in dark brown by Ki67 immunohistochemistry were the proliferative cells Immunohistochemistry ×400 Fig.7 In SHEE cell culture, the nucleus stained red or pink by PI was dead cell, the green nucleus was living cell Fluorescent ×400 Fig.8 The cell labeled by TdT was apoptotic cell in which the chromatin of nucleus condensed in block, a pyknotic nucleus in the upper right conner was seen TdT labeled ×400

图1 SHEE培养在盖坡片上，活细胞单层生长，胞浆较丰富，细胞核大小一致，有核仁×400 图2 SHEE培养细胞细胞核椭圆形，核仁较大，胞浆有较丰富的线粒体和张力原纤维EM ×10 000 图3 SHEE细胞呈球状，有伪足贴壁，表面有密集微绒毛SEM ×5 000 图4 同上细胞贴壁，呈星状或多角形，有丰富伪足和胞浆突，核区隆起有较多微绒毛SEM ×5 000 图5 SHEE细胞染色体仍属二倍体Giemsa染色×1 000 图6 SHEE细胞Ki67免疫组织化学染棕黄色为增殖细胞×400 图7 SHEE培养细胞出现死细胞，胞核和胞浆PI染色呈红色或淡红色，蓝色细胞核为活细胞荧光显微镜×400 图8 细胞TdT标记阳性为凋亡细胞，染色质凝集呈块状，右上角有一固缩细胞核TdT标记×400

The testis index, testis volumes were same as the annual changes of testis mass. The curves of annual variation were all unimodality.2 The spermatogenetic cycle of Myospalax cansus comprises seven stages with significant features: Stage I , from February to April, the testis were at the stage of spermatogonia proliferation. In this period, testis index and the number of spermatogonia began to rise. Other spermatogenic cells had not yet formed; Stage II to III, from March to April, primary spermatocyte meiosis period. The testis index was highest in this stage, and spermatogenic cells were in spermatocyte stage, the primary spermatocyte meiosis generated to secondary spermatocyte; Stage IV, from April to May, spermatocytes continued to split, germ cells appeared in seminiferous tubules; Stage V, in May, sperm formation, spermatids of seminiferous tubules were transformed to spermatozoa, a large number of sperms existed in the lumen; Stage VI, spermatozoa emission period, from May to June, testis index were a significant drop and mature spermatozoa excluded gradually; VII, the testicular activity ceased basically from July to September, November to January of the following year, the spermatogenic activity ceased completely. Therefore, Myospalax cansus are animals of seasonal reproduction, spermatogenesis cycle is discontinuous type.

睾丸系数、体积和重量的年周期变化规律一致，变化曲线呈现单峰型。2甘肃鼢鼠雄性生殖腺的年周期活动由7个特征明显的时期构成：Ⅰ期，2～3月份，精原细胞增殖期，睾丸系数开始上升，精原细胞进行有丝分裂，其他生精细胞尚未形成；3～4月份为Ⅱ～Ⅲ期，初级精母细胞成熟分裂期，睾丸系数达到最大，生精细胞大多处于精母细胞阶段，初级精母细胞减数分裂生成次级精母细胞；Ⅳ期，4～5月份精母细胞继续进行分裂，精细胞在生精小管内出现；Ⅴ期，5月份，精子形成期，曲细精管中精细胞变态成精子，在管腔中存在大量的精子；Ⅵ期，精子排放期，5～6月份，睾丸系数显著下降，成熟精子从生精小管上脱离，逐渐排除；Ⅶ期，精原细胞停滞期，7～9月份睾丸生精活动基本停滞，11～翌年1月，生精活动完全停止。

Study the bioactivity of the n-HA/PA66 composite and the effects it would be to body's metabolism of calcium and phosphorus ion in vivo.(3) Study the osteo-conductivity and the ability to repair bone defect of the porous n-HA/PA66 composite and the feasibility use it as the scaffold of bone tissue engineering. Objects and Methods as follows: 1.To evaluate the biocompatability of nano-hydroxyapatite crystals and polyamide composite (n-HA/PA66) with the L929 cells.To proceed the morphological observation and take pictures of L929 cells after 1d,2d,4d,and 7d of co-cultured with extract of n-HA/PA66 ,and direct contact with n-HA/PA66.To determine light absorbtion value of every hole under 500 nm with enzyme linked immunity instrument after 1 d,2 d,4 d,and 7 d of contact of n-HA/PA66 extract with L929 cells,and direct contact with n-HA/PA66.In the meanwhile calculate the relative multiplication rate of cells,and evaluate them by six degree tests for cytotoxicity. To investigate the acute and chronic toxic reaction on the whole body induced by the new nano-hydroapatite crystals and polyamide composite(n-HA/PA66)after implanting in vivo and its effects on partial constitution of animal organs after implanting in vivo,and evaluate the potential and degree of subcuticular stimulation reaction.

本实验主要由以下三部分组成：一、n-HA/PA66 复合材料在动物体内、体外的生物相容性及生物安全性评价二、n-HA/PA66 复合材料植入动物体内的生物活性及近期对机体钙、磷代谢影响的实验研究三、网孔 n-HA/PA66 复合材料作为支架修复兔桡骨节段缺损的动物实验研究主要研究目标及方法如下：参照 GB/T16886.5-1997-ISO 10993-5：1992《医疗器械生物学评价细胞毒性试验体外法》之评价标准和要求，采用规定的 L929 细胞(小鼠结缔组织成纤维细胞)，分别经直接接触和材料浸提液与细胞共培养等方式对 n-HA/PA66 复合材料进行细胞毒性测试，采用细胞形态观察法观察两种细胞各组在 24h、48h、72h、5 天后各时相点的细胞形态学变化，并在显微镜下照相，从而对细胞与材料的生物相容性进行定性评价；同时采用细胞生长抑制法，以酶标仪定量测定评价各组 1，2，4，7 天 L929 细胞的相对增殖率，以定量测定并判别材料对细胞的毒性程度。

Of CBMC can be purified by Mini-MACS as CD34〓 stem cells. B. The number of CD34〓 stem cells can expand to 40.24±9. 86 fold after 14 days. C. No matter in the expression of CD1a, CD80, CD86, and HLA-DR, or in the function of stimulating xenogenous lymphocyte proliferation, there was no difference between CD34+ stem cell DCs or monocyte DCs. D. The percentage of CD3〓CD56〓 cells is the same in CIK cells co-culture with DCs transfected with SKOV3 RNA, CIK cells co-culture with DCs, and CIK cells. E. The expansion rate of CIK cells can be accelerated by co-culturing with loaded or unloaded DCs. However, the expansion rate between loaded or unloaded group is the same. F. The strongest cytotoxicity against SKOV3 cell line was achieved by CIK cells co-cultured with DCs loaded with SKOV3 RNA.

结果：1、Mini-MACS分选系统可自CBMC中提取0.78±0.31%的CD34〓细胞；2、体外培养14天后可获得原始CD34〓细胞量40.24±9.86倍的细胞；3、不论在CD1a、CD80、CD86及HLA-DR的表达上，或是刺激异体淋巴细胞增生的功能上，脐带血CD34〓细胞与单核细胞来源的DC都没有差别；4、CIK细胞中CD3〓CD56〓双阳性的表达率在与RNA转染DC共培养的CIK细胞组、与DC共培养的CIK细胞组及单纯CIK细胞组3组间比较无差异；5、脐带血CIK细胞增殖显著，培养14天时可扩增18.18±5.59倍，培养21天时可扩增35.02±6.30倍；5、与未转染或转染DC共培养的CIK细胞在培养第14天后增殖速率大于单纯CIK细胞。

Our study includes four aspects. In the first aspect we study several important conditions of porcine oocytes maturation in vitro and oocytes cleavage after parthenogenetic activation and found mNCSU-23+15IU/mlPMSG+20IU/mlHCG+15% PFF+0.57mMcysteine is a good culture condition .When the Cocs are cultured in it ,the maturation rate and oocytes cleavage rate are higher than those of foreign covered. Our result are (86.7±3.35)% and (86.3±4.16)% and the highest report of foreign is(85.7±4.1)%.In the second aspect we study the effect of different chemical activations on development of porcine parthennogenetic embryo and found two best activation method. The first one is that putting the maturation MII oocytes in the 20μmol/L ionomycin for 30 minutes and then putting them in the NCSU-23 condition containing 5μg/mICB and 5mM/L6-DMAP for 3.5 hours, the oocytes cleavage rate and morulae/blastocysts development rate are (76.7±7.6)% and (37.1±6.4)%.The second one is that putting the maturation MII oocytes in the 200μM/L Thimerosal for 20 minutes and then putting them in the NCSU-23 condition containing 8mM DTT for 30 minutes

本研究分为4个部分，第一部分对影响猪卵母细胞体外成熟和孤雌激活后胚胎分裂的几个重要条件进行了比较研究，确立了一种较好的培养方法：与颗粒细胞共培养，找到了一种适合猪卵母细胞体外成熟的培养基：mNCSU-23+15IU/mlPMSG+20IU/mlHCG+15%PFF+0.57mM半胱氨酸，成熟率和分裂率分别为(86.7±3.35)%和(86.3±4.16)%，国外报道的最高成熟率为(85.7±4.1)%；第二部分对猪卵母细胞孤雌激活的化学方法进行了研究，确立了化学激活猪卵母细胞的两种最佳方法：1将成熟的去卵丘颗粒细胞的MII期卵母细胞用20μmol/Lionomycin作用30min，再将卵母细胞培养于含5μg/mlCB和5mM/L 6-DMAP(6-二甲基氨基嘌呤)的NCSU-23培养液中，卵裂率和桑囊胚发育率达到(76.7±7.6)%和(37.1±6.4)%2将成熟的去卵丘颗粒细胞的MII期卵母细胞在200μM/L的Thimerosal中处理20min，再与8mM的DTT共孵育30min，卵裂率和桑/囊胚形成率为(81.0±2.8)%和(39.6±2.7)%；第三部分对孤雌激活胚胎的培养条件进行了研究，确立了一种最佳的胚胎培养条件：在SOF简单培养基中添加颗粒细胞进行前3天的培养，然后转入添加胎牛血清的NCSU—23培养基并和输卵管上皮细胞进行后期的培养，其桑椹胚和囊胚的发育率为(59.5±3.2)%；第四部分研究了IGF-I

1、Cur inhibits K562 cells growth and induces cell apoptosis may be correlated with the down-regulation of p210~、inhibition of protein tyrosine phosphorylation and the signaling molecules such as p-Erk1/2、c-myc which are relevant with cell growth and apoptosis; 2、Cur synergizes STI571 to inhibit K562 cell growth and induce cell apoptosis may be correlated with the down-regulation of p210~、inhibition of protein tyrosine phosphorylation and the signaling molecules such as Hsp90、PKC which are relevant with cell growth and apoptosis; 3、Cur reverses the resistance of K562/G01 cells to STI571, and synergizes STI571 to inhibit K562/G01 cell growth and induce cell apoptosis; 4、Cur inhibits human originated CML CD34~+ cell growth、induces cell apoptosis, and enhances STI571 to down-regulate the expression of p210~, finally inhibit cell growth and induce cell apoptosis.

从以上实验结果我们得出如下结论： 1、Cur抑制K562细胞增殖、诱导细胞凋亡的作用可能与其下调p210~、蛋白酪氨酸磷酸化水平以及抑制下游p-Erk1/2、c-myc等信号分子有关； 2、Cur协同STI571抑制K562细胞增殖、诱导细胞凋亡的作用可能与其下调p210~、蛋白酪氨酸磷酸化水平以及抑制Hsp90和下游PKC等信号分子有关； 3、Cur可逆转K562/G01细胞对STI571的耐药性，并与STI571协同抑制K562/G01细胞增殖和诱导凋亡，其抑制K562/G01细胞增殖、诱导细胞凋亡的作用可能与其下调p210~、蛋白酪氨酸磷酸化水平以及抑制下游Procaspase-3和NF-κB等信号分子有关； 4、Cur可抑制来源于CML患者骨髓的CD34~+细胞的增殖并诱导其凋亡，还可协同STI571下调CML CD34~+细胞p210~表达，进而协同抑制细胞增殖、诱导细胞凋亡。

I didn't watch TV last night, because it .

昨晚我没有看电视，因为电视机坏了。

Since this year, in a lot of villages of Beijing, TV of elevator liquid crystal was removed.

今年以来，在北京的很多小区里，电梯液晶电视被撤了下来。