阅读说明：本技术 一种诱导人脐带间充质干细胞向神经细胞方向分化的方法 (Method for inducing differentiation of human umbilical cord mesenchymal stem cells towards nerve cells ) 是由 张蕾 赵荷艳 于 2021-08-24 设计创作，主要内容包括：本发明公开了一种诱导人脐带间充质干细胞向神经细胞方向分化的方法,包括以下步骤：人脐带间充质干细胞的分离与培养；将人脐带间充质干细胞按1×10～(4)个/孔的密度接种于24孔培养板,用细胞培养液培养过夜；将24孔培养板中的细胞培养液换为诱导分化液,继续培养14d,期间每隔3d换液一次。本发明通过应用脑源性神经营养因子(BDNF)胶质细胞源性神经营养因子(GDNF)明显诱导脐带间充质干细胞分化为神经细胞。(The invention discloses a method for inducing human umbilical cord mesenchymal stem cells to differentiate towards nerve cells, which comprises the following steps: separating and culturing human umbilical cord mesenchymal stem cells; human umbilical cord mesenchymal stem cells are arranged in a 1 x 10 mode 4 Inoculating the culture plate with the density of each hole in a 24-hole culture plate, and culturing the culture plate with a cell culture solution overnight; the cell culture medium in the 24-well plate was changed to the differentiation-inducing medium, and the culture was continued for 14 days, during which time the medium was changed every 3 days. The method obviously induces the umbilical cord mesenchymal stem cells to be differentiated into the nerve cells by applying the brain-derived neurotrophic factor (BDNF) and the glial cell-derived neurotrophic factor (GDNF).)

1. A method for inducing differentiation of human umbilical cord mesenchymal stem cells towards nerve cells is characterized by comprising the following steps:

s1, separating and culturing human umbilical cord mesenchymal stem cells;

s2, culturing the human umbilical cord mesenchymal stem cells obtained in the step S1 according to the ratio of 1 x 10⁴Inoculating the culture plate with the density of each hole in a 24-hole culture plate, and culturing the culture plate with a cell culture solution overnight; the cell culture solution comprises the following components: 89ml of DMEM/F12 basal culture solution, 10ml of FBS and 1ml of 100 XPS;

s3, changing the cell culture solution in the 24-hole culture plate into an induced differentiation solution, and continuously culturing for 14d, wherein the solution is changed every 3 d; the components of the differentiation inducing liquid are as follows: 97ml of DMEM/F12 basal medium, 2ml of FBS, BDNF2ug, 2ug of GDNF and 1ml of 100 XPS.

2. The method for inducing differentiation of human umbilical cord mesenchymal stem cells towards nerve cells according to claim 1, wherein the method for isolating and culturing human umbilical cord mesenchymal stem cells of step S1 comprises:

s11, acquiring human umbilical cords from a caesarean section in a hospital, and acquiring informed consent of a premenstrual and puerperal woman;

s12, rinsing the umbilical cord in phosphate buffered saline for 30 seconds on a super clean bench, cutting the umbilical cord into sections with the length of 2-3cm, peeling off umbilical cord blood vessels by using forceps, and shearing Wharton's gum into pieces with the length of 0.5-1mm by using an ophthalmic scissors³A size tissue mass;

s13, uniformly sticking the tissue blocks to 75cm²Culturing at the bottom of the bottle, wherein the tissue blocks are separated by about 5mm, adding a small amount of cell culture fluid to prevent the tissue blocks from floating, and standing at 37 deg.C and 5% CO₂Culturing in an incubator;

s14, after overnight, adding 10ml of cell culture solution into a culture bottle, and continuing to culture;

s15, after 5-7d, cells can grow out around the tissue block, and at the moment, the cell culture solution is replaced;

s16, changing the solution every 2-3 days, after about 7-9 days, paving the bottom of the bottle with cells about 80%, digesting with 0.25% pancreatin, and carrying out passage, wherein after 3 passages are carried out, the cells can be used for subsequent experiments.

3. The method for inducing differentiation of human umbilical cord mesenchymal stem cells towards nerve cells as claimed in claim 2, wherein the cell culture fluid in steps S13, S14 and S15 comprises: 89ml of DMEM/F12 basal medium, 10ml of FBS and 1ml of 100 XPS.

4. The method for inducing differentiation of human umbilical cord mesenchymal stem cells towards neural cells according to claim 1, wherein the umbilical cord of step S11 is cultured in cells within two hours after obtaining.

5. The method for inducing differentiation of human umbilical cord mesenchymal stem cells toward neural cells according to claim 1, wherein the 24-well culture plate of step S2 is incubated with poly-L-lysine at a concentration of 0.01%.

Technical Field

The invention belongs to the technical field of cell culture, and particularly relates to a method for transdifferentiating human umbilical cord mesenchymal stem cells to nerve cells.

Background

The important pathological basis of neurodegenerative diseases is local neuron degeneration and necrosis, and the current main method for treating the diseases is to supplement deficient neurotransmitters by medicaments, but the problem of corresponding neuron degeneration and necrosis cannot be fundamentally solved, and the cell replacement therapy is increasingly the research hotspot of the treatment method of the diseases. Mesenchymal stem cells are a class of pluripotent stem cells developed from mesoderm, exist in connective tissues and organ mesenchyme, and can differentiate into three layers of embryonic cells under specific conditions. The mesenchymal stem cells existing in Wharton's jelly and perivascular tissues of the fetal umbilical cord of a mammal theoretically have the characteristics of stem cells such as self-renewal, proliferation and multidirectional differentiation, are rich in source, have no ethical constraint in application, and have low immunogenicity, so that the human umbilical cord mesenchymal stem cells can be a good source for treating cells of nervous system diseases, but the primary problem is to confirm whether the human umbilical cord mesenchymal stem cells have the potential of differentiating towards nerve cells.

The authors cultured human umbilical cord Wharton's glial-derived mesenchymal stem cells in vitro in research, successfully induced the differentiation of umbilical cord mesenchymal stem cells towards nerve cells by improving the culture environment, confirmed that human umbilical cord mesenchymal stem cells have the potential of transdifferentiation towards nerve cells, and provided a certain experimental basis for the replacement therapy of nervous system diseases by human umbilical cord mesenchymal stem cells.

Disclosure of Invention

The invention mainly aims to provide a method for inducing the transdifferentiation of human umbilical cord mesenchymal stem cells to nerve cells.

The invention adopts the following technical scheme for solving the technical problems:

a method for inducing human umbilical cord mesenchymal stem cells to differentiate towards nerve cells comprises the following steps:

s1, separating and culturing human umbilical cord mesenchymal stem cells;

s2, culturing the human umbilical cord mesenchymal stem cells obtained in the step S1 according to the ratio of 1 x 10⁴Inoculating the culture plate with the density of each hole in a 24-hole culture plate, and culturing the culture plate with a cell culture solution overnight; the cell culture solution comprises the following components: 89ml of DMEM/F12 basal culture solution, 10ml of FBS and 1ml of 100 XPS;

s3, changing the cell culture solution in the 24-hole culture plate into an induced differentiation solution, and continuously culturing for 14d, wherein the solution is changed every 3 d; the components of the differentiation inducing liquid are as follows: 97ml of DMEM/F12 basal medium, 2ml of FBS, BDNF2ug, 2ug of GDNF and 1ml of 100 XPS.

Further, the method for isolating and culturing human umbilical cord mesenchymal stem cells of step S1 comprises:

s11, acquiring human umbilical cords from a caesarean section in a hospital, and acquiring informed consent of a premenstrual and puerperal woman;

s12, rinsing the umbilical cord in phosphate buffered saline for 30 seconds on a super clean bench, cutting the umbilical cord into sections with the length of 2-3cm, peeling off umbilical cord blood vessels by using forceps, and shearing Wharton's gum into pieces with the length of 0.5-1mm by using an ophthalmic scissors³A size tissue mass;

s13, uniformly sticking the tissue blocks to 75cm²Culturing at the bottom of the bottle, wherein the tissue blocks are separated by about 5mm, adding a small amount of cell culture fluid to prevent the tissue blocks from floating, and standing at 37 deg.C and 5% CO₂Culturing in an incubator;

s14, after overnight, adding 10ml of cell culture solution into a culture bottle, and continuing to culture;

s15, after 5-7d, cells can grow out around the tissue block, and at the moment, the cell culture solution is replaced;

s16, changing the solution every 2-3 days, after about 7-9 days, paving the bottom of the bottle with cells about 80%, digesting with 0.25% pancreatin, and carrying out passage, wherein after 3 passages are carried out, the cells can be used for subsequent experiments.

Further, the cell culture solution in the steps S13, S14 and S15 comprises: 89ml of DMEM/F12 basal medium, 10ml of FBS and 1ml of 100 XPS.

Further, the umbilical cord of step S11 is subjected to cell culture within two hours after the obtaining.

Further, the 24-well plate in step S2 is incubated with poly-L-lysine at a concentration of 0.01%.

Advantageous effects

The method obviously induces the umbilical cord mesenchymal stem cells to be differentiated into the nerve cells by applying the brain-derived neurotrophic factor (BDNF) and the glial cell-derived neurotrophic factor (GDNF).

Drawings

FIG. 1 is a schematic diagram of the morphological change of human umbilical cord mesenchymal stem cells after induced differentiation.

Wherein panel A is the cell morphology after 1 day of induced differentiation when the cells are in the form of long fusiform characteristic of mesenchymal stem cells; panel B shows the cell morphology 7 days after induced differentiation, when there were cells that had appeared rounded and had small protrusions (indicated by arrows) at both ends; panels C and D show the morphology of cells after induced differentiation for 14 days, when the cells grew unipolar, bipolar or multipolar processes (arrows) similar to nerve cells from round bodies.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are illustrative only for the purpose of explaining the present invention, and are not to be construed as limiting the present invention. Example (b):

the materials used in the present invention include:

(1) dulbecco's Modified Eagle Media (DMEM)/F12 basal medium (Thermo Co.);

(2) fetal Bovine Serum (Fetal bone Serum, FBS, Gibco Corp.);

(3) brain-derived neurotrophic factor (brain-derived neurotrophic factor, BDNF, Invitrogen);

(4) glial cell-derived neurotrophic factor (glial cell-derived neurotrophic factor, GDNF, Invitrogen);

(5) 0.25% pancreatin (Sigma);

(6)100 Xpenicillin Streptomycin mixed solution (Penicilin and Streptomyces, PS, Beyotime Co.);

(7) Poly-L-lysine (Poly-L-lysine, PLL, Sigma): dd H₂Preparing a diluent with the final concentration of 0.01% by using O, filtering, and subpackaging and storing;

(8) cell culture solution: 89ml of DMEM/F12 basal culture solution, 10ml of FBS and 1ml of 100 XPS;

(9) inducing differentiation liquid: 97ml of DMEM/F12 basal medium, 2ml of FBS, BDNF2ug, 2ug of GDNF and 1ml of 100 XPS.

A method for inducing human umbilical cord mesenchymal stem cells to differentiate towards nerve cells comprises the following steps:

s1, separating and culturing human umbilical cord mesenchymal stem cells:

s11, taking the umbilical cord after the fetus is delivered in the caesarean section, and immediately placing the umbilical cord in sterile PBS (the umbilical cord must be subjected to cell culture within two hours after the umbilical cord is obtained);

s12, rinsing the umbilical cord in PBS for 30 seconds on a super clean bench, cutting into sections with the length of 2-3cm, cutting off amnion, peeling off umbilical cord blood vessels with forceps, and shearing Wharton's jelly into pieces with the length of 0.5-1mm with ophthalmologic scissors³A size tissue mass;

s13, uniformly sticking the tissue blocks to 75cm²Culturing at the bottom of the bottle, wherein the tissue blocks are separated by about 5mm, adding a small amount of cell culture fluid to prevent the tissue blocks from floating, and standing at 37 deg.C and 5% CO₂Culturing in an incubator;

s14, after overnight, adding 10ml of cell culture solution into a culture bottle, and continuing to culture;

s15, after about 5-7 days, cells can grow out around the tissue blocks, the cell culture solution is replaced once, and most of the tissue blocks can be removed during the solution replacement;

s16, changing the solution every 2-3 days, after about 7-9 days, paving the bottom of the bottle with cells about 80%, digesting with 0.25% pancreatin, and carrying out passage, wherein after 3 passages are carried out, the cells can be used for subsequent experiments.

S2, inducing the human umbilical cord mesenchymal stem cells to transdifferentiate into nerve cells:

s21, incubating a 24-hole culture plate with poly-L-lysine, and removing the culture plate after overnight;

s22, the human umbilical cord mesenchymal stem cells are expressed by 1 multiplied by 10⁴Inoculating the culture medium into 24-well culture plates incubated by poly-L-lysine at a density of one well/well, and culturing the culture medium overnight;

s23, changing the cell culture solution in the 24-hole culture plate into an induced differentiation solution, and continuing to culture for 14 days, wherein the induced differentiation solution is changed every 3 days.

As shown in fig. 1, wherein panel a is a cell morphology 1 day after induced differentiation, when the cell is in a long fusiform shape peculiar to the mesenchymal stem cell; panel B shows the cell morphology 7 days after induced differentiation, when there were cells that had appeared rounded and had small protrusions (indicated by arrows) at both ends; fig. C and D show cell morphology after induced differentiation for 14 days, in which cells grew unipolar, bipolar or multipolar processes (arrows) similar to nerve cells from round cell bodies, which are significantly different from the original long fusiform form, indicating that human umbilical cord mesenchymal stem cells can differentiate toward nerve cells after induced differentiation.