A method for separation, purification and culture of naked mole-rat Leydig cells

Abstract

The invention relates to the field of cell culture, in particular to a method for separating and purifying naked mole-rat testicular interstitial cells, which adopts enzyme digestion and a Percoll continuous density gradient method to separate and purify naked mole-rat testicular interstitial cells. The method of the present invention can obtain a large number of naked mole rat testicular stromal cells with normal functional activity in a simple, efficient and economical manner, and the culture under hypoxic conditions can ensure that the cells can still maintain their biological properties in an in vitro environment. characteristics, so that it is convenient to further study the special physiological functions of naked mole rat Leydig cells directly in a pure in vitro cell culture model, thus providing an important theoretical basis for exploring the biological mechanism and applying it to clinical related fields.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to a method for separating, purifying and culturing Leydig cells of naked mole rat testes. Background technique [0002] Leydig cells are distributed in the loose connective tissue between the testicular seminiferous tubules, accounting for 2% to 4% of the cells in all testicular tissues, and their main function is to synthesize and secrete testosterone, which accounts for 95% of total plasma testosterone (Dobashi M, Fujisawa M, Yamazaki T, et a1. Inhibition of steroidogenesis in Leydig cells by exogenous nitric oxide Occursindependently of steroidogenic acute regulatory protein (star) mRNA [J]. ): 203-209.). In view of the important role of Leydig cells, a set of stable methods for isolating, purifying, and primary culturing Leydig cells, and maintaining the activity of the cells secreting testosterone, is necessary for further research on the functions of Leydig cells. is o...

AI Technical Summary

Problems solved by technology

However, current international research is limited to phenotype research, and the mechanism of low testosterone levels in naked mole rats has not yet been studied. Once the biological mechanism is clarified, it will have broad application prospects in the field of clinical medicine, especially for men. The prevention and treatment of infertility diseases is of great significance

[0004] Regarding the primary culture of mesenchymal cells, Klinefelter et al. (Klinefeher GR, Hall PF, Ewing LL. Effect of luteinizing hormone deprivation in situ onsteroidogenesis of rat Leydig cells purified by a muhistep procedure [J]. BiolReprod, 1987) are generally used at home and abroad. , 36(3):769-783.) introduced the method, but because the method requires harsh experimental conditions, it is difficult for general laboratories to achieve

At present, there is no research on the in vitro culture method of naked mole rat Leydig cells

The main reasons are as follows: 1) naked mole rat is a kind of temperature-changing animal, the temperature of its somatic cells in vitro culture needs to be explored, and its unsteady metabolic rate makes its somatic cell in vitro culture medium different from ordinary rats or mice ; 2) There is a certain degree of functional overlap between the testis and epididymis of naked mole rats, and the testis also serves as a place for storing sperm while producing sperm, which brings certain difficulties to the separation of interstitial cells; 3) Naked mole rats The proportion of interstitial components in the testis is high, and the separation method of interstitial cells is different from that of other rodents; 4) Naked mole rats have adapted to the external hypoxic environment, so the oxygen concentration in the somatic cell culture environment is different from that in normoxia. animals that live in the environment and require groping

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