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Study of the Effects of Soy Isoflavone on the Osteoblast in Vitro

Author: FanZuoZuo
Tutor: WangXueQian
School: Tianjin Medical University
Course: Biochemistry and Molecular Biology
Keywords: Osteoblasts Soy isoflavones Phytoestrogens Estradiol Postmenopausal osteoporosis
CLC: Q813.1
Type: Master's thesis
Year: 2007
Downloads: 111
Quote: 2
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Abstract


Objective 1. And the perfect OB separation and culture technology 2. Explore the SI to provide a scientific basis for the prevention and treatment of PMO SI vitro culture in all aspects of the proliferation and differentiation of primary rat OB. Method 1. Separation of the in vitro rat OB Primary culture and identification: 24 hours from newborn Wistar rats skull, using a number of digestion were separated OB alternately adding trypsin and collagenase type II in primary cultured in vitro, in cultured 5th days, 7 days, 10 days, Wright's staining and ALP staining (azo coupling Act) identification OB. 2. SI on the proliferation and differentiation of in vitro primary rat OB research: (1) dose groups: the the experimental divided into normal control group (Normal Control group), the the SI concentration group (10 -5 M 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M), positive The control group (E 2 group the 10 -10 M E 2 ). (2) SI OB proliferation, differentiation: When the cells adherent change dosing medium were measured dosing medium 1-7 days cells MTT (OD), and cultured for 2 days and 4 days later to take the cell culture the liquid measurement OB extracellular ALP and BGP content. 3. SI rat OB expression of collagen type I: to be adherent cells, for the dosing medium and cultured for 4 days after the extraction of total cellular RNA, RT-PCR amplification of collagen type I cDNA simultaneous amplification of the housekeeping gene β-actin as an internal control. Scanning PCR products electrophoresis images, calculate the ratio of the relative abundance of collagen type I / β-actin projections relative expression levels of collagen type I gene. Results 1. The separation of the in vitro rat OB and primary culture and identification: The cultured cells were cells elongated fusiform, triangular forms cytoplasmic processes of various shapes, showing clusters growth and the typical the OB morphological characteristics; The ALP staining positive (positive rate of> 95%), the identification of the OB. 2. SI result of proliferation and differentiation in vitro primary rat OB: (1) the different concentration SI role in OB, MTT results show: compared with the normal control group, 1 to 4 days in culture,, SI10 -8 M SI10 -5 M group MTT (OD) values ??were significantly higher (P <0.05), slowed from the first five days proliferation, proliferation of 2,3 days most with the E 2 group showed no significant (P> 0.05); SI10 -9 M group from the first three days MTT (OD) value is lower than the normal control group, a statistically significant (P <0.05). The proliferation of normal control group the curve visible incubation period of 4-7 days, 1-4 days in a period of exponential growth, SI concentration group E 2 group into the exponential proliferation phase earlier than the normal control group ; E 2 , SI10 -8 M, SI10 -6 M, SI10 -5 M group their proliferation curve presents a step-like. (2) ALP measurement results: Compared with the normal control group, OB cultivate the second day of the group E 2 and SI10 -6 M group can significantly improve the OB secretion ALP activity (P <0.05); OB cultured for 4 days, E 2 group SI10 -6 M group and SI10 , -5 M group significantly improve OB secretion ALP activity (P <0.05). E 2 group SI10 -5 M group, SI10 -9 M group ~~ SI10 -7 M The active the group OB secretion of ALP lower than E 2 group (P <0.05). (3) BGP measurement results: Compared with the normal control group, OB cultivate the second day of the group E 2 and SI10 -7 M group can significantly increase the activity of the the OB secretion BGP (P <0.05). OB cultured for 4 days, E 2 group SI10 -8 M group and SI10 -7 M group can significantly improve the OB secretion ALP activity (P <0.05). E 2 group, the SI10 -9 M group, SI10 -8 M group, SI10 -6 M group and SI10 -5 M group OB secretion BGP activity below E 2 group (P <0.05). 3. SI rat OB expression of collagen type I: Compared with control group, the by SI10 -7 M SI10 -5 M group E 2 The group can be a significant increase in the OB type I collagen synthesis (P <0.05). Conclusion 1. Newborn 24 hours Wistar rat skull material trypsin and type II collagen using alternating join several digestive Separation OB, high purity can be obtained in larger quantities function active in OB. 2. SI OB proliferative activity by incubation time and SI concentration: effective concentration range of SI, OB proliferation, 10 -8 the M ~~ 10 -5 M significant, and the most active days 2,3; low concentration SI10 -9 M 3 day MTT (OD) value lower than the normal control group. The role of each concentration of Si OB, proliferation curves showing different shapes and also different for each cycle time limit may be prompted at different concentration range, the different reactions of different cells in the proliferation cycle of effective ingredient, while Description SI promote cell proliferation The complexity and diversity. 3. SI10 -6 M and 10 -5 SM to improve OB secretion ALP activity; S110 -8 M 10 -7 M improve the content of the OB outside BGP. 4. SI10 -7 M, SI10 -5 M group can increase the expression of collagen type I mRNA. 5. SI and E 2 OB proliferation and differentiation is consistent with a role in promoting bone formation.

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