The Cultivation Of Rat Hypothalamic Cells And The Empirical Study Of The Secretion Of β-endorphin In Vitro

Objective:Drug addiction has become the most popular problem in social and medical area. It has been considered as a chronic disease like hypertension, asthma and non-insulin dependent diabetes, because there are so many similar factors among them such as the heredity, recurrent attacks and the treatment. Recently it has been found that the endorphin secreted by neuron inhibited for the use of opium. Once the external opium was removed, the body may have the psychological and physical symptom for the endorphin below the normal level. With the development of cell graft, the activity cells implanted the body, and the implanting cells, like a "biological pump", can secrete endorphin constantly. So it can replenish the deficient of endorphin in drug withdraw body. That may be an ideal treatment for drug addiction. The rat hypothalamic cells were studied in this experiment. According to characters of hypothalamic cells, the cultivation of hypothalamic cells was established. Observing the cells growing, mature and ageingwith histological and immunocytochemistry method. The function of secreting endorphin of cultivating cells was evaluated with ELISA. Resuscitated the cells after half month freezing keep, and compared with the morphous and the function of endorphin secretion of the cultivating cells between two periods. Method:The hypothalamic cells were isolated and purified by trypsin digestion. The neuron were identified with neuron-specific enolase (NSE) immunocytochemical staining, and observed the cells changes at different culture time. Had cultured the hypothalamic cells for 9 days, and changed the culture liquid every 3 days, collected the culture liquid every day, the concentration of endorphin was assaied with ELISA. Furthermore, 9 days later, reculture the cell after trypsin digestion, and assaied the concentration with ELISA as above. Resuscitated the cells after half month freezing keep, and then observing the cells with histology and immunocytochemistry method. Culturing the reanimated hypothalamic cells for 9 days, and changed the culture liquid every 3 days, collected the culture liquid every day, the concentration of endorphin was assaied with ELISA. Result:1, cultivation and identification of hypothalamic cells: (1) Cellular growth and shape: on the first day of cultivation, the cellular body was slightly dilation. On the 7th day,cellular structure was intact, the boundary was regularand the chromaffin granules were clear. But on the ninthday, it retained at the same level. (2) Immunocytochemical staining: The NSE positive cellscan be identified with hypothalamic cells. 2, Assaied the endorphin, in the culture liquid.(1) There was no difference of the secrete of endorphin among every day in the 9 days of cultivation. The mean concentration of endorphin was 43.83±3.17 pmol/1 F=0.644p=0.734(2) There was no difference among every 3 days for changing of the liquid in the 9 days of cultivation. F=0.009p=0.991(3) There was no difference between the before and after trypsin digestion (F=1.724, p=0.066).3,There was no difference on the morphous and immunocytochemistry and the function of secrete of endorphin of freezing keeping and resuscitated hypothalamic cells, compared with primary cultivation. Conclusions:1, By trypsin digestion and disperse cultivation of the rat hypothalamic cells, we can get stable healthy growing cells which have similar structure and immunocytochemistry with the cells in the body.2, On the first day of cultivation, the rat hypothalamic cells can secrete endorphin and up to a stable level. With the time...