Inducer for inducing differentiation of mesenchymal stem cells into estradiol-secreting cells

Abstract

The present disclosure belongs to the field of biological medicines, and relates to an inducer for inducing differentiation of mesenchymal stem cells into estradiol-secreting cells. The inducer for inducing differentiation of mesenchymal stem cells into estradiol-secreting cells uses human mesenchymal stem cell serum-free culture medium as a substrate and comprises the following components in mass concentration ratios: 20-60 mg/L of bone morphogenetic protein-4, 20-60 mg/L of bone morphogenetic protein-7, 2-8 mg/L of retinoic acid, 2-8 mg/L of resveratrol, 2-8 mg/L of icariin, 2-8 g/L of benzamide, 2-8 g/L of chloroplatinic acid hexahydrate, 2-8 g/L of ethanolamine, 2-10 g/L of erythropoietin and 2-10 g/L of vascular endothelial growth factor. The inducer for inducing differentiation of mesenchymal stem cells into estradiol-secreting cells provided by the present disclosure has a high induction efficiency.

Claims

1. An inducer for inducing differentiation of mesenchymal stem cells into estradiol-secreting cells, wherein the inducer comprises the following components in mass concentration ratios: 20-60 mg/L of bone morphogenetic protein-4, 20-60 mg/Lof bone morphogenetic protein-7, 2-8 mg/L of retinoic acid, 2-8 mg/L of resveratrol, 2-8 mg/L of icariin, 2-8 ug/L of benzamide, 2-8 ug/L of chloroplatinic acid hexahydrate, 2-8 ug/L of ethanolamine, 2-10 ug/L of erythropoietin and 2-10 ug/L of vascular endothelial growth factor.

2. The inducer for inducing differentiation of mesenchymal stem cells into estradiol-secreting cells according to claim 1, wherein the inducer comprises the components in the following mass concentration ratios: 50 mg/L of bone morphogenetic protein-4, 50 mg/L of bone morphogenetic protein-7, 8 mg/L of retinoic acid, 6 mg/L of resveratrol, 6 mg/L of icariin, 4 ug/L of benzamide, 6 ug/L of chloroplatinic acid hexahydrate, 4 ug/L of ethanolamine, 5 ug/L of erythropoietin and 5 ug/L of vascular endothelial growth factor.

Description

DETAILED DESCRIPTION OF THE EMBODIMENTS

(1) Unless specially stated, experimental methods in the following examples are all conventional methods. Instruments and reagents used in the experiment are commercially available.

Example 1

(2) An inducer for inducing differentiation of mesenchymal stem cells into E2-secreting cells in this example comprised the following components in mass concentration ratios: 40 mg/L of BMP4, 50 mg/L of BMP7, 8 mg/L of RA, 2 mg/L of resveratrol, 6 mg/L of icariin, 4 g/L of benzamide, 8 g/L of chloroplatinic acid hexahydrate, 4 g/L of ethanolamine, 10 g/L of EPO and 2 g/L of VEGF. The above components were successively added into a human mesenchymal stem cell serum-free culture medium (or DMEM+10% FBS or other types of commercially available mesenchymal stem cell culture mediums) in the mass concentration ratios, evenly mixed and filtered to remove bacteria.

(3) The various components of the inducer of the present disclosure were all commercially available products: a human mesenchymal stem cell serum-free culture medium with brand LONZA, and article number 00190632; BMP4 with brand Gibco, and article number PHC9533; BMP7 with brand Gibco, and article number PHC7204; RA with brand Sigma, and article number R2625; resveratrol with brand Sigma, and article number R5010-100 MG; icariin with brand Shanghai Microcrystalline Biology, and articule number 489-32-7; benzamide with brand Sigma, and article number 135828; chloroplatinic acid hexahydrate with brand Sigma, and article number 206083; ethanolamine with brand Sigma, and article number 8008490100; EPO with brand PeproTech, and article number CYT-201; VEGF with brand PeproTech, and article number 96-100-20-2.

Example 2

(4) An inducer for inducing differentiation of mesenchymal stem cells into E2-secreting cells in this example comprised the following components in mass concentration ratios: 50 mg/L of BMP4, 20 mg/L of BMP7, 2 mg/L of RA, 2 mg/L of resveratrol, 2 mg/L of icariin, 4 g/L of benzamide, 6 g/L of chloroplatinic acid hexahydrate, 4 g/L of ethanolamine, 8 g/L of EPO and 8 g/L of VEGF. The above components were successively added into a human mesenchymal stem cell serum-free culture medium (or DMEM+10% FBS or other types of commercially available mesenchymal stem cell culture mediums) in the mass concentration ratios, evenly mixed and filtered to remove bacteria.

Example 3

(5) An inducer for inducing differentiation of mesenchymal stem cells into E2-secreting cells in this example comprised the following components in mass concentration ratios: 50 mg/L of BMP4, 50 mg/L of BMP7, 8 mg/L of RA, 6 mg/L of resveratrol, 6 mg/L of icariin, 4 g/L of benzamide, 6 g/L of chloroplatinic acid hexahydrate, 4 g/L of ethanolamine, 5 g/L of EPO and 5 g/L of VEGF. The above components were successively added into a human mesenchymal stem cell serum-free culture medium (or DMEM+10% FBS) in the mass concentration ratios, evenly mixed and filtered to remove bacteria.

Example 4

(6) By example of human adipose mesenchymal stem cells, the effect of the inducer of the present disclosure on inducing differentiation of mesenchymal stem cells into E2-secreting cells was illustrated. The 3 generations of human adipose mesenchymal stem cells were passaged and inoculated into a 96-well plate in an amount of 110.sup.4/cm.sup.2. When almost 80% of cells were fused and vigorously gre, differentiation was induced. The induction conditions are seen in Table 1.

(7) TABLE-US-00001 TABLE 1 Induction condition groups Group Inducer Control Human mesenchymal stem cells serum-free culture medium (no inducer) group Human mesenchymal stem cells serum-free culture medium + BMBP4 (blank) 50 mg/L + BMP7 50 mg/L Induction group 1 Induction Human mesenchymal stem cells serum-free culture medium + BMBP4 group 2 50 mg/L + BMP7 50 mg/L + RA 8 mg/L Induction Human mesenchymal stem cells serum-free culture medium + BMBP4 group 3 50 mg/L + BMP7 50 mg/L + RA 8 mg/L + resveratrol 6 mg/L + icariin 6 mg/L Induction Human mesenchymal stem cells serum-free culture medium + BMBP4 group 4 50 mg/L + BMP7 50 mg/L + RA 8 mg/L + resveratrol 6 mg/L + icariin 6 mg/L + benzamide 4 g/L Induction Human mesenchymal stem cells serum-free culture medium + BMBP4 group 5 50 mg/L + BMP7 50 mg/L + RA 8 mg/L + resveratrol 6 mg/L + icariin 6 mg/L + benzamide 4 g/L + chloroplatinic acid hexahydrate 6 g/L Induction Human mesenchymal stem cells serum-free culture medium + BMBP4 group 6 50 mg/L + BMP7 50 mg/L + RA 8 mg/L + resveratrol 6 mg/L + icariin 6 mg/L + benzamide 4 g/L + chloroplatinic acid hexahydrate 6 g/L + ethanolamine 4 g/L Induction Human mesenchymal stem cells serum-free culture medium + BMBP4 group 7 50 mg/L + BMP7 50 mg/L + RA 8 mg/L + resveratrol 6 mg/L + icariin (present 6 mg/L + benzamide 4 g/L + chloroplatinic acid hexahydrate 6 g/L + disclosure) ethanolamine 4 g/L + EPO 5 g/L + VEGF 5 g/L

(8) The morphological change of cells during the induction was carefully observed. At the induction time of 1-9 days, a culture solution was taken from each group and then centrifuged at 2000r/min for 10 min, and the supernatant was stored at 20 C., subsequently the E2 content was respectively detected and statistically analyzed. The E2 content was detected following operations specified by the instruction of an estradiol ELISA kit (Shanghai Ongke Biotechnology Co., Ltd). The results are seen in Table 2.

(9) TABLE-US-00002 TABLE 2 Comparison of induction results of different inducers. E2 secretion (pg/ml)(n = 5, x s) After After After After After After After After After Group 1 day 2 day 3 day 4 day 5 day 6 day 7 day 8 day 9 day Control 3.1 0.3 3.4 0.1 2.9 0.2 3.4 0.1 4.1 0.2 5.1 0.3 5.2 0.3 5.5 0.1 5.6 0.1 group Induction 25.2 1.0 43.8 1.4 52.6 2.7 61.6 3.6 71.6 4.6 79.2 4.4 91.6 3.7 89.6 1.9 94.6 4.6 group 1 Induction 27.8 1.5 47.5 2.1 97.4 3.3 117.9 22.0 127.3 12.5 163.6 13.7 187.4 9.2 259.5 213 280.0 12.9 group 2 Induction 44.0 2.3 96.9 11.5 144.5 7.8 189.8 7.6 260.0 1.7 365.5 11.0 374.3 23.4 477.7 22.1 510.3 11.9 group 3 Induction 46.8 2.4 123.3 9.7 189.9 23.4 288.0 22.9 349.1 9.3 469.3 18.5 533.3 32.6 627.7 21.7 680.4 23.5 group 4 Induction 55.7 9.4 123.9 14.6 207.4 12.5 326.0 21.8 425.9 21.1 530.4 29.7 617.7 28.4 719.3 24.5 813.4 17.8 group 5 Induction 83.2 2.6 132.2 13.2 217.2 21.1 319.2 17.0 432.2 18.9 632.2 23.8 791.2 19.6 823.2 32.3 932.2 28.5 group 6 Induction 87.2 2.5 196.2 11.5 278.2 12.7 387.2 13.8 513.2 22.1 687.2 21.9 878.2 19.6 941.2 27.5 1187.2 32.8 group 7

(10) It can be seen from the induction results in Table 2 that the induction of human adipose mesenchymal stem cells using the inducer of the present disclosure has the highest efficiency, and the cells obtained by induction secrete the most content of E2. After the inducer of the present disclosure is added, E2 is continuously secreted after 2 days, and the secretion gradually increases. The maximum daily secretion of E2 can be reached about 5-7 days after induction, and E2 can be continuously secreted.