The Researches Of Mechanical-biological In Chondrocytes With Conditional Knockout Indian Hedgehog

Objective:Compared with normal cells, to analyze Ihh gene knockout mice source cartilage cells in vitro culture in the process of mechanical, biological characteristics, clear Ihh gene knock out to maintain and promote the seed cell biology characteristic advantage.Methods:1、newborn Ihhfl/fl pure merge with Col2a1- Cre ERt2 genetically engineered mice 30,were randomly divided into experimental group and control group, experimental group(Tamoxifen), intraperitoneal injection of TM, intraperitoneal injection of the control group in the same amount of vegetable oil injection for five days in a row, acute digestive rib cartilage cells.2 、 real-time PCR detection Ihh gene expression, analysis Ihh gene knockout efficiency.3 、 CCK 8 test and flow cytometry respectively at the same time points in the experimental group and control group rib cartilage cell proliferation and apoptosis(n =10).4、RT-PCR analysis Col I、Gli- 1, Col II, Col X, Agg and Mmp13 m RNA expression level change(n = 5).(5) the microtubules sucking and semi-infinite body cell mechanics model and technology Ihh knockout rib cartilage cells after the change of mechanical properties(n = 5).Results:1、Detection of CCK 8 found that knockout Ihh gene, the cartilage cell proliferation efficiency is relatively lower than control, including 48 h, 72 h is most significant, 72 nm wavelength absorbance, respectively(0.534 ±0.15),(0.722±0.13)(p < 0.05) and(0.758 ±0.14),(0.946±0.17)(p < 0.05).2、The flow cytometry test analysis found that: 3 days, 5 days, 7 days of cell early apoptosis rate than the control group were increased(P < 0.05).3、Rt-pcr analysis of Col II and Agg m RNA expression level 4.2 times higher than the control group respectively(P < 0.01) and 3.2(P < 0.05), Col X and Mmp13 m RNA expression level 3.6 times, respectively(P < 0.01) and 2.6(P < 0.05).4 、 Microtubules sucking and semi infinite body mechanics model of measuring mechanical properties of cartilage cells found: instantaneous modulus modulus(E0),equilibrium modulus(E∞) and apparent viscosity(u) are high relative to the control group(P < 0.05).Conclusions:Conditional knockout chondrocytes after Ihh gene can effectively improve the Col II and Agg expression, inhibition of Col X and Mmp13 expressed. Mechanical properties and cartilage cells increased obviously. Therefore, knock out Indian hedgehog gene for optimal seed cells has a certain value.