tmem16a as a marker of osteoporosis and its application, osteoporosis diagnostic kit and medicine
A diagnostic kit and osteoporosis technology, applied in the field of disease markers, can solve the problems of inability to compare with the true value, inaccurate judgment, and high measurement cost
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Embodiment 1
[0051] In this example, the expression of TMEM16A in osteoclasts of patients with osteoporosis and normal people was measured, and a small amount of bone tissue was extracted from patients with osteoporosis through minimally invasive surgery, and primary osteoclasts were isolated from them, using complete medium (DMEM-α medium: fetal bovine serum = 9: 1) for cell culture. Through PCR experiment, analyze the change of TMEM16A RNA content in osteoclasts of osteoporosis patients compared with normal people. Using TMEM16A antibody to carry out cell immunoblotting experiments, comparative analysis of TMEM16A expression in osteoclasts from patients with osteoporosis and changes in normal expression. The result is as figure 1 As shown, it was found that the expression of TMEM16A in the bone tissue of osteoporosis patients was about 1.5 times higher than that of normal people, so the increased expression of TMEM16A is a marker of osteoporosis.
Embodiment 2
[0053] In this implementation, a mouse model of osteoporosis was first established, and then the content of TMEM16A in bone tissue of osteoporotic mice was measured.
[0054] The establishment method of the mouse osteoporosis model: select female C57 mice aged 3-6 months, and artificially remove the ovaries of the female mice through surgery, and culture them normally for four weeks to build a model, simulating the osteoporosis caused by estrogen deficiency in postmenopausal women. The male mice were male C57 mice aged 2-4 months. The testicles of female mice were artificially removed by surgery, and they were cultured normally for four weeks to build a model, simulating the senile osteoarthritis caused by the sharp decrease in the secretion of androgen in elderly men. mass porosis.
[0055] The measurement methods for the expression of TMEM16A mainly include: measuring the change of RNA content of TMEM16A by PCR, mainly including: extraction of total RNA by Trizol method, rev...
Embodiment 3
[0058]In this example, osteoclasts were induced to differentiate in vitro. The osteoclast induction and differentiation method was as follows: bone marrow cells were extracted from the bone marrow of C57 mice at 6-8 weeks, and the erythrocyte lysate was fully lysed and then centrifuged to extract bone marrow stromal cells. Add 10ng / After 24 hours, the supernatant was taken out, centrifuged at 1500rpm, and the culture medium containing 50ng / ml Rankl and 30ng / ml M-CSF was added to continue to cultivate for 5 days, during which the medium was replaced once, and it was used after 5 days TRAP staining kit was used to detect whether osteoclasts were successfully induced. The expression level of TMEM16A was detected by PCR and immunoblotting experimental methods, and the experimental methods were the same as those in the above-mentioned examples.
[0059] Then detect the content of TMEM16A in the cells after normal induction 1, 3 and 5 days respectively, the results are as follows: ...
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