| Background and Objective:Recently, the incidence of heart disease is increasing annually, the world rate of 2%. Myocardial infarction, MI is the one of diseases which effect human health mostly, while the chronic heart failure, CHF is the main reason of MI death. Molecular bypass, also named as therapeutic angiogenesis, provides a new cure for recovery of injured cardiac muscle cells and rebuilding of blood vessel. It is approved by the ten-year research that molecular bypass is useful to build the effective side cycle, decrease the blood-leaking symptom radically and improve the patients' situation. Molecular bypass becomes the hot topic of the world heart-blood physic now. So, study of the inner mechanism of molecular bypass and development of the medicine of molecular bypass will help the patients who suffer blood-leaking patients to improve the quality of life and prolong the length of life. Our team is always commit ourselves to research Morinda officinalis oligosaccharides, MOO's function of protect the heart. A preliminary study of the early confirmed:MOO has obvious mitigate hypoxia ischemia reperfusion injury of the myocardial cells and may contribute to chick urinary bladder and rat myocardial ischemia, increase its microvascular (MVD) quantity and density; significant upward regulate angiogenesis dependent VEGF, bFGF protein expression. Angiogenesis gene chip results show MOO increases a variety mRNA expression of angiogenic gene reduce suppressor gene expression of angiogenesis. MOO dose-dependent improve TRPC6 protein expression in vascular cells. Therefore, this experiment in human umbilical vein endothelial cells (HUVEC) aimed to study angiogenesis effect of MOO further by the vascular endothelial cell migration and tubular experimental to observe the effect of MOO on hypoxia injury endothelial cell migration and lumen-like structure of formation, using the flow cytometry technology to observe the hypoxia injury groups of cell proliferation. And then, we expected to provided a more intuitive evidence that MOO plays a part in ischemic myocardium protection and in ischemic heart establish collateral promotion.Materials and Methods:1 HUVEC cultureHUVEC in 50ml culture flasks by adding 10% FBS RPMI1640 containing high glucose medium, at 37℃,5% CO2, saturated humidity incubator 1～2d, cells gradually grow into a single fusion-like. Nucleus is observed under inverted microscope, round, membrane clear, multilaterally-shaped spindle-shaped cells, showing a typical paving stone-like. When the culture bottles are covered with about 80%, cells can be digested with trypsin passaged or cryopreserved.2 Cell model of production and determineUsing oxygen scavenger hydrosulfite cause anoxia, kind of plate after the training, each hole 24h dumped medium, with no sugar Earle's liquid detergent cell 2, normal control group join Medium 1640; model groups joined with sodium hydrosulfite final concentration of 1 mm without sugar Earle's fluid, causing cell hypoxia, positive drug control group join tablets (2 g·L-1), the Group joined with MOO hydrosulfite final concentration of 1 mm without sugar Earle's fluid and MOO (0.45 g·L-1), medium (0.15 g·L-1), small (0.05 g·L-1),37°c; incubating 4h culture plates, suction to remove abandoned various experimental hole liquid, Earle with sugar-free's liquid detergent cell 2, various experimental holes are replaced with drug serum-free medium 1640, continue to cultivate 12h, causing cell reoxygenation.3 Experimental groupThis experiment is divided into six groups: Normal cellsHypoxia-reoxygenation injury model groupHypoxia-reoxygenation injury model plus positive control Shexiangbaoxin Pill (2g/L)Hypoxia-reoxygenation injury model plus small dose of MOO group (0.05g/L)Hypoxia-reoxygenation injury model plus medium-dose group of MOO group (0.15g/L)Hypoxia-reoxygenation injury model plus high-dose group of MOO group (0.45g/L)Observed indicatorsHUVEC cells in vitro are cultured normally with 3-8 generations to produce hypoxia-reoxygenation injury model, given different doses of Morinda sugar chain drug intervention 12h,①Inverted microscope observation of the growth of state;②Cytometry various experimental cell growth of the groups;③hypoxia conditions group human umbilical vein endothelial cell migration status;④observation of hypoxia in the conditions group human umbilical vein endothelial cells and the formation of small tubes.Statistical processingData were analyzed by Spss 16.0 statistical processing software and demonstrated with mean±standard deviation(x±s), the comparison between groups adopted single-factor analysis of variance, a=0.05 was considered as the significant test level. When there are significant differences between groups,compare with q inspection.Result1 Model injuries for 4h, showing that cell contraction changesgarden and shape irregular, the gap became larger, and some fall off under the microscope. Positive control group and each MOO dose groups could promote cell proliferation, and increase with the dose increased.2 Flow cytometry results:compared with the normal group, hypoxia/reoxygenation injury model group increased by G2+s-phase cells; Compared with the model group, high- and medium-dose group of MOO has weakened this trend, and the cells is similar in morphology to normal cells, but effect in small-dose group of MOO is not obvious.3 Compared with hypoxia/reoxygenation injury model group, cell migration of all MOO groups is clearly increasing, and canaliculization is a dose-dependent manner.Conclusion1 MOO can exert significant protective effects against hypoxia/reoxygenation injury in HUVEC, Inhibit apoptosis, Protect effectively vascular endothelial cells.2 MOO can significantly promote vascular endothelial cell migration and canaliculization. This is its role to promote therapeutic angiogenesis as one of the important foundation. |
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