Calmodulin-dependent protein kinase (CaMK), activated by auto-phosphorylation at the presence of calcium and calmodulin, is widely distributed in eukaryotes. CaMKs are important mediators of calcium signal in eukaryotes. Recent researches have suggested that CaMKⅡ is involved in the regulation of meiotic cell cycle of oocytes. It plays functional roles in meiotic maturation, polar body extrusion, fertilization and egg activation. As one of the down-stream signaling molecules of calcium, CaMKⅡ facilitates the inactivation of maturation promoting factor (MPF) and cytostatic factor (CSF) following fertilization, as well as the spindle microtubule organization and centrosome duplication. Although the functions of CaMKⅡ in oocyte meiosis are versatile and essential, the present results are primarily obtained from low vertebrates and mouse. In future studies, the function and regulation of this kinase in other mammals should be stressed.

AIM: To study the changes in capillarity of skeletal muscle during acclimation to high altitude, and explore the effects of a certain extent physical activity under hypoxia on capillary formation and the role of vascular endothelial growth factor (VEGF) in this process. METHODS: 48 Wistar rats were divided into 3 groups: Ⅰ normoxic control; Ⅱ hypoxia and Ⅲ hypoxia+exercise. Rats of Ⅱ and Ⅲ groups were subjected to hypobaric hypoxia for 5 weeks (23 h/d). They were first brought to simulated 4 000 m altitude, where rats of the Ⅲgroup were forced to swim for 1 h/d (6 d/week). Then the animals were ascent to 5 000 m. Biomicrosphere method was used to determine blood flow of skeletal muscle. The mean fiber cross-sectional area (FCSA), capillary density (CD) and capillary/fiber ratio (C/F) of red portion of the lateral head of the gastrocneminus were assayed by myofibrillar ATPase histochemistry. VEGF and its receptor KDR were assayed with immunohistochemistry method.RESULTS: By comparison with the normoxic control, 5-week hypoxic exposure resulted in a decrease in cross-sectional area of skeletal muscle fiber and an increase in CD, but the C/F remained unchanged. The blood supply to the gastrocnemius was not changed. After 5-week-exercise at high altitude, the muscle fibers did not undergo atrophy. CD, C/F, and the blood flow at rest increased significantly. VEGF protein was found primarily in the matrix between muscle fibers; KDR were shown mainly in endothelial cells of capillary. VEGF was more strongly stained in the skeletal muscle of hypoxia-exercise rats.CONCLUSION: Hypoxia itself can not induce neovascularization. While exercise during hypoxic exposure can lead to capillary formation. VEGF and KDR may play roles in it. New capillary formation benefits the blood supply, oxygen delivery and working performance at high altitude.

The cytoplasmic and nuclear changes during the development of goat oocytes were studied with light and electron microscopy. The oocyte development may be divided into 8 stages in accordance with the number and distribution of follicle cells and the size of the follicle. The results indicated that the Golgi complex, mitochondria, smooth endoplasmic reticulum and cortical granules became well developed and they moved into the cortical region as the oocyte development proceeded. In the oocytes in the follicle of 1.5-3mm in diameter, all the mitochondria became hooded and the number and size of fibrillar centers in the nucleolus reached maximum, but the nucleolar compaction still not occured at this stage. By the time when the follicle reached 3.5-5mm in diameter, the follicular cell processes began to degenerate, the microvilli of the oocyte withdrew from the zona pellueida and the oocytes might be cultured to mature in vitro. In the mature oocytes, Golgi complex and rough endoplasmic reticulum disappeared, the cortical granules arranged themselves in one layer beneath the oolemma, the mitochondria dispersed in the central region of cytoplasm and the eggs were ready for fertilization.

Objective To investigate distribution of integrain ?1 in human oocytes and embryos before and after maturation/fertilization,dring the first and second meiosis,in morulae and blastocyst stage. Methods Human oocytes and embryos were stained by anti-integrin ?1 (PharMingen 09351D) and FITC-labeled second antibody (PharMingen 110094D),and observed by confocal microscope. Results Integrin ?1 concentrated in the nuclear area in matured oocytes,zygotes and 2-cell stage embryo,indicating that integrin ?1 might involve in mitosis.Dislike mouse,there was no distribution of integrin ?1 on the well-accepted sperm binding area on the oocytes,indicating that integrin ?1 might not involve in the binding and fusion of human sperm to human oocytes.Conclusion It was first postulated that integrin ?1 might actively involve in the pronuclear fusion process in human in vitro fertilized oocytes.The marker of ectotrophoblasts,polarized distribution of integrin ?1, appeared at the morulae stage.The stronger expression in ectotrophoblasts around the inner cell mass might indicate some specific function of the ectotrophoblasts near the ICM.

Objective To comparatively study the features of two reprogramming systems of induced pluripotent stem cells (iPSCs)from human dental origin.Methods Two kinds of reprogramming system,i.e.STEMCCA lentivirus /feed layer and Sen-dai virus /matrigel were used to induce human stem cells from apical papilla(SCAP)into iPSCs,respectively.The induction efficien-cies,workload of generating iPSCs,aneuploidy karyotype ratio,complexities of eliminating exogenous transcription factors and spe-cific markers expression were compared between these two systems.Results The STEMCCA reprogramming system required to prepare the feeder cell MEF.The reprogramming efficiency was 0.1%.Transcription gene-free iPSCs cells were obtained by the Cre-loxp enzyme digestion technique at the later stage.Sendai virus reprogramming system was feeder-free and the preparation of matrigel was quite simple with unified standard.The reprogramming efficiency was 0.7%,which was much higher than that of STEMCCA system(P <0.05).The exogenous virus and transgenes could be gradually eliminated after several passages of natural subclone.Conclusion The Sendai virus/matrigle reprogramming system is much more applicable for the induction of iPSCs from dental origin than the STEMCCA system.

Objective To investigate the inhibitory effects of RNA interference (RNAi) on the expression of matrix metalloproteinase-2 (MMP-2) gene and growth, adhesion,invasiveness and migration of ovarian cancer cells. Methods One specific target sequence of MMP-2 gone and one non-specific sequence (NC group) were chosen,the medium DMEM as blank group.After transfection of ovarian cancer OVCAR-3 cells, the RT-PCR and Western blot were used to detect mRNA and protein expression of MMP-2 gene, the growth ability was detected by MTT assay, the abilities of adhesion was detected by cell adhesion assay, the invasion and migration were detected by Matrigel invasion assay and wound healing assay. Results By contrast to the NC group,the mRNA expression was decreased by 73.8 ％,78.8 ％ and 78.4 ％(P＜ 0.05) in 24 h,48 h and 72 h after transfection and protein expression was decreased by 72.6 ％,81.2 ％ and 76.4 ％(P＜ 0.05) respectively at the same time. The 48 h group had the most efficient inhibitory effect. Cell growth curve revealed that cell growth was not significantly inhibited (P＞ 0.05). Adhesion was significantly reduced,the inhibitory rate was 55.0 ％ at 60 min and 44.8 ％ at 90 min (P＜ 0.05),respectively. Invasion and migration were significantly reduced as well,the inhibitory rate on invasion and migration were 29.7 ％ and 35.8 ％(P＜0.05), respectively. Conclusion siRNA mediated MMP-2 down-regulation in ovarian OVCAR-3 cells can inhibits its adhesion,invasion and migration,but do not significantly affect its growth,suggesting a important target to ovarian cancer gene-therapies.

Objective: To study the role of vascular endothelial growth factor (VEGF) during angiogenesis in oral squamous cell carcinoma (SCC).Methods:Surgical specimens from 45 patients with oral SCC were examined for VEGF expression and microvessel density(MVD) by immunocytochemical staining. Anti-VEGF polyclonal antibody was used to determine VEGF expression and anti-CD 34 monoclonal antibody was used to determine MVD. Results:①VEGF protein was mainly found in cytoplasm of cancer cells and in few endothelial cells. Stained microvessels were mainly distributed in the regions of the connective tissue near the cancer nest;②there was a close positive correlation between MVD and VEGF expression;③SCC with high MVD or high VEGF level was mostly found with metastasis in cervical lymph node (P

Objective To evaluate the skill and outcome of arthroscopic reconstruction of posterior cruciate ligament (PCL) with retention of PCL remnant. Methods From April 2004 to June 2006, 38 patients (38 knees) with PCL deficiency were verified by clinical and arthroscopic examinations. Of them, there were 9 knees combined with disruption of the posterolateral comer, 6 with rupture of the posteromedial corner, 8 with lateral meniscus tear and 4 with medial meniscus tear. With reservation of PCL remnant and synovium, all the impaired PCLs were reconstructed with single bundle of autogenous quadrupled hamstring tendons under arthroscopy. Interference screws were used for direct anatomic fixation of the reconstructed ligament. Results No severe comphcations occurred at early stage after operation in all 38 patients who were followed up for 12-37 months (average 20.79 months). Lysholm score was improved significandy from 40-70 points (mean 51.32 pints) before operation to 70-100 pints (mean 92.37 points) at the latest follow up (t=-30.14, P<0.01). According to International Knee Documentation Committee (IKDC) score, there was a remarkable improvement from 16 abnormal knees (grade C) and 22 severely abnormal knees (grade D) preoperatively to 18 normal knees ( grade A), 18 nearly normal knees (grade B) and 2 abnormal knees at the latest follow up (Z=-6.00, P <0.01). Of 38 patients, 36 returned to normal sports level but 2 degraded level of sports. Conclusions Arthroscopic PCL reconstruction with retention of PCL remnants is a feasible technique, with satisfactory outcome. Preservation of PCL remnants and synovium may be beneficial to biological incorporation and reinnervation of the reconstructed ligament.

AIM: To study the changes in myocardial blood flow (MBF), capillarization and cardiac function in the rat during acclimation to hypoxia. METHODS: Myocardial capillary density (CD) and capillary/myocyte ratio (C/M) was assayed by alkaline phosphatase histochemistry. Biomicrosphere method was used to determine MBF in the rat after 5, 15 or 30 days hypobaric hypoxic exposure (5 000 m). RESULTS: In the course of hypoxia, MBF and cardiac function increased in the right ventricle. However, in the left ventricle, acute hypoxia caused an increase in MBF and a decrease in cardiac function. Both returned to the control level on continued hypoxic exposure. Neovascularization occurred after 15 day or 30 day of hypoxic exposure in both ventricles, judged from the significant increment of C/M ratio albeit the CD remained unchanged in the right ventricle. CONCLUSION: Our findings indicate that adaptive changes in rat heart during acclimation to hypoxia include: ① persistent increase in MBF, hypertrophy associated with increase in capillarity and enhanced cardiac function of the right ventricle; ② increase in MBF and depression of cardiac function at first, then followed by recovery of MBF and increase in capillarity accompanied with recovery of left ventricular function.