CN113801843B - Method for enhancing human urine-derived stem cell stem property - Google Patents
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Abstract
The invention discloses a method for enhancing the dryness of human urine-derived stem cells (hUSCs). The Boldine (Boldine) with the final concentration of 1.5-3 mu M is added into the basal culture medium of the hUSCs, so that the expression of OCT4 genes in the in-vitro long-term subculture process of cells can be effectively promoted, the expression of MYC genes in the hUSCs can be effectively promoted by the 3 mu M Boldine, the dryness of the hUSCs is enhanced, the problems of reduced cell viability, easy aging and differentiation and the like in the in-vitro culture of the hUSCs are avoided, and the in-vitro research and transformation application of the hUSCs are promoted.
Description
Technical Field
The invention relates to a method for enhancing the stem cell stem property of human urine source.
Background
The stem cells have higher application value in cell therapy and drug screening. Human urine-derived stem cells (hUSCs) are cells which are separated from human urine, have good proliferation activity and multidirectional differentiation potential, are wide in source, convenient to obtain, safe and noninvasive, can realize self-renewal and clonal growth in vitro under a certain culture environment, highly express mesenchymal stem cell surface markers such as CD44, CD73, CD90, CD105, CD146 and the like, can differentiate into cells of multiple germ layers sources such as kidney cells, bone cells, nerve cells, vascular endothelial cells and the like under a special induction environment, and play an important role in tissue engineering of urinary systems, motor systems, nerve systems and disease treatment [1-2] . However, the efficiency of isolated culture of hUSCs in vitro is low, and as the number of subcultures increases, the cell stem gradually weakens, and partial senescence or differentiation occurs, which greatly restricts the intensive research and clinical application of hUSCs [3] 。
At present, the research on the dry regulation of hUSCs at home and abroad is relatively few, chun et al culture hUSCs under the condition of hypoxia by utilizing a hUSCs culture medium containing collagen (ColI), and the discovery that the ColI can effectively improve the proliferation and differentiation capacity, the chromosome stability and the dry gene expression level of cells [4] The method comprises the steps of carrying out a first treatment on the surface of the Wu et al studied the dry regulation effect of long-chain non-coding RNAKCNQ1DN on hUSCs, and the result shows that the knock-down of KCNQ1DN expression in hUSCs can promote cell proliferation and up-regulate expression of related transcription factors c-Myc, nanog and Rex1 mRNA and protein [5] . Compared with protein intervention and gene knockout, the small molecular compound has stable structure and low costThe characteristics of simple operation and the like play important roles in regulating and controlling the biological behaviors of stem cells in vitro [6] While research results on small molecule compounds to enhance the dryness of hccs in vitro have not been reported.
Cell antioxidant damage is an effective way to enhance stem cell stem property and delay stem cell aging [7] . Inappropriate culture conditions or modes can lead to the reduction of expression level of stem cells such as OCT4, MYC, hTERT and other stem genes, so that the stem cells gradually age or differentiate [8] . In the long-term in-vitro culture process of human urine-derived stem cells hUSCs, the partial pressure and the oxygen content of oxygen in a culture medium can be gradually increased, and the hUSCs in a high-oxygen environment are extremely easy to generate oxidative damage under the action of oxygen free radicals, so that the hUSCs have adverse effects of cell aging, dry weakening and the like. Therefore, the realization of the stem cell antioxidation injury culture is beneficial to enhancing the stem property of the hUSCs and delaying the cell aging, so that the hUSCs can keep higher cell activity for a long time. The research shows that the isoquinoline alkaloid Boldine (figure 1) has strong antioxidant activity, and has good cytoprotective effect by demonstration of the antioxidant activity at the level of cells and animal models [9] . However, the research of the Boerdines in the stem cell field is relatively few at present, and the research of the high antioxidant activity of the Boerdines on the stem property of the hUSCs is favorable for further widening the pharmacological action range of the Boerdines, and simultaneously is favorable for developing medicines for enhancing the stem property of the hUSCs and delaying the aging of the hUSCs, enhancing the in vitro long-term subculture capability of the human adult stem cells such as the hUSCs and improving the clinical application value of the hUSCs [10] 。
Disclosure of Invention
The invention aims at providing a method for effectively enhancing the stem cell stem property of human urine source.
Collecting 200-300 mL of sterile urine of healthy adults, centrifuging at normal temperature, and separating and culturing the hUSCs by using a self-made simplified hUSCs basal medium.
And (3) identifying the basic biological characteristics of the hUSCs, observing the cell morphology by an inverted phase contrast microscope, identifying the cell surface markers by a flow cytometry method, and measuring the cell viability by a tetramethyl thiazole blue (MTT) method.
The identification shows that the hUSCs with normal cell morphology and good cell activity are obtained, and the effective enhancement of the dryness of the hUSCs is realized by adding 1.5-3 mu M Boerdines into a basic culture medium.
The detection of the dryness level of the hUSCs after the Bohr alkali action is verified from two aspects of cell morphology and molecular biology characteristics, namely, the change of the cell morphology after the Bohr alkali action is observed by an inverted microscope, namely, the change of the mRNA expression of OCT4 and MYC genes of cells after the Bohr alkali action is detected by a real-time fluorescent quantitative PCR technology (real time quantitative reverse transcription PCR, RT-qPCR) with different concentrations (containing 1.5 mu M and 3 mu M).
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The foregoing and/or additional aspects and advantages of the invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings, in which:
FIG. 1 shows the chemical structural formula information of the small molecule compound Bohr's base;
FIG. 2 shows the growth of P2 generation hUSCs in human urine derived stem cell basal medium according to one embodiment of the invention;
FIG. 3 shows the results of the identification of mesenchymal stem cell surface markers CD44 and CD90, hematopoietic cell surface marker CD34, endothelial cell surface marker CD31 antigen expression of P3 generation hUSCs, according to one embodiment of the invention;
FIG. 4 shows the results of experiments on the effect of human urine-derived stem cell culture medium with varying concentrations of Bohr's alkali on the viability of P3 generation hUSCs cells, according to one embodiment of the invention;
FIG. 5 shows the results of experiments on the effect of human urine-derived stem cell culture medium supplemented with various concentrations of Bohr's alkali on the dry gene expression of P3 generation hUSCs, according to one embodiment of the invention;
FIG. 6 shows the results of experiments on the effect of human urine derived stem cell medium supplemented with 1.5. Mu.M, 3. Mu.M Boerdines on the expression of the dry gene of P7 generation hUSCs, according to one embodiment of the invention;
FIG. 7 shows the growth of P7 generation hUSCs in human urine-derived stem cell basal medium and human urine-derived stem cell medium supplemented with 1.5. Mu.M, 3. Mu.M Boldine, according to one embodiment of the invention.
Detailed Description
Embodiments of the present invention are described in detail below. The embodiments described below by referring to the drawings are illustrative only and are not to be construed as limiting the invention.
Example 1
Experiments were performed to investigate the culture growth of hscs in human urine derived stem cell basal medium according to the following procedure:
primary culture of hscs was performed using human urine derived stem cell basal medium consisting of KSFM, ham's F, HG-DMEM, FBS, hEGF, ITS and penicillin-streptomycin, subcultured in vitro to P2 generation for subsequent cell experiments.
The cell viability experiment was performed by inoculating P2 generation hscs with good growth status, and the cell morphology was kept as "long fusiform" with clonal adherent growth (fig. 2).
Example 2
Experiments were performed to identify cell surface markers of hscs and to determine their biological characteristics with mesenchymal stem cells, according to the following procedure:
flow cytometry is used for identifying cell surface antigen expression of P3 generation hUSCs, hUSCs single cell suspension is prepared by using hUSCs basal medium containing serum, after normal temperature centrifugation, resuspended cells are washed by using PBS buffer solution and cell density is adjusted, after split charging into an EP tube, 1 mug of each of mouse anti-human antibodies CD31-PE-Cy7, CD34-PE, CD44-FITC and CD90-APC is added for light-shielding incubation for 30min, after incubation, 300g of the cells are centrifuged for 5min, unlabeled cells are washed by using PBS buffer solution, the suspension is repeated for 1 time, 200 mug of PBS is added for resuspension cells after supernatant is discarded for membrane filtration, and the filtered cell suspension is detected by using a flow cytometer.
The flow cytometry identification result shows that the positive rate of CD31 of the P3 generation hUSCs is 0.84%, the positive rate of CD34 is 0.98%, the positive rate of CD44 is 96.23%, and the positive rate of CD90 is 88.67% (figure 3), which suggests that the hUSCs hardly express the surface markers of endothelial cells and hematopoietic cells, but highly express the surface markers of mesenchymal stem cells.
Example 3
Experiments were performed to study the effect of adding varying concentrations of boldine on the viability of the hccs cells, and to determine the optimal boldine addition concentration, according to the following procedure:
MTT method for measuring cell viability of P3 generation hUSCs, preparing single cell suspension after digestion of hUSCs by pancreatin, inoculating the single cell suspension into 96-well plates (4 multiple wells are arranged) at a cell density of 1000 cells per well, respectively feeding Bohr's alkali into the cells at a concentration of 0.3 mu M, 1.5 mu M, 3 mu M, 15 mu M and 30 mu M, continuously observing for 72h, adding 100 mu L MTT solution every day at fixed time, incubating for 4h, dissolving and oscillating for 10min with 150 mu L DMSO, and detecting absorbance value (OD 570) at 570nm by an enzyme-labeled instrument.
MTT results showed that the cell morphology remained unchanged and improved the cell viability of hUSCs after 24, 48 and 72 hours of induction of hUSCs by 1.5-3. Mu.M Bohr's base, peaking the cell OD at a concentration of 1.5. Mu.M (FIG. 4).
Example 4
Experiments were performed to study the effect of adding varying concentrations of boldine on the expression of the dry gene of hscs and to determine the optimal boldine addition concentration according to the following procedure:
the RT-qPCR method for detecting the dry gene expression level of P3 generation hUSCs, inoculating hUSCs into 6-well plate (3 multiple wells are arranged) at the cell density of 1000 cells per well, administering Boerdine to the cells at the same concentration as above, continuously observing for 72h, extracting total RNA respectively, and performing reverse transcription to synthesize cDNA, and the RT-qPCR method for detecting the dry gene OCT4 and MYC expression level (beta-actin is an internal reference gene) by adopting 2 -ΔΔCt Statistical analysis was performed on the experimental results, and the primer sequences are shown in Table 1.
TABLE 1 primer information
RT-qPCR results show that the Bohr alkali (1.5-3 mu M) can obviously up-regulate the expression level of the hUSCs dry gene OCT4, and the 3 mu M Bohr alkali has a certain promotion effect on the expression of the hUSCs dry gene MYC, which suggests that the Bohr alkali can enhance the dryness of the hUSCs to a certain extent (figure 5).
Example 5
Experiments were performed to investigate the effect of human urine derived stem cell medium supplemented with 1.5 μm, 3 μm boldine on P7 generation hscs dry gene expression according to the following procedure:
RT-qPCR results show that the dryness of the hUSCs gradually weakens with the increase of the times of the successive generations, and both the B1 (1.5 mu M) (hUSCs-P7-B1) and the B2 (3 mu M) (hUSCs-P7-B2) of the Boehringer's alkali dosing group can enhance the OCT4 gene expression of the P7 generation hUSCs (hUSCs-P7) of the control group without significant influence on the expression of MYC genes (figure 6); after the P7 generation hUSCs are cultured in the human urine-derived stem cell basal medium for a period of time, the cell morphology is gradually changed from a long spindle shape into a round shape or a fiber shape, and part of cells begin to age or differentiate, while the cells are synchronously cultured in the human urine-derived stem cell culture medium containing 1.5 mu M and 3 mu M Boermannine, so that the cell morphology is kept well and the cell growth activity is good (figure 7).
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
While embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that: many changes, modifications, substitutions and variations may be made to the embodiments without departing from the spirit and principles of the invention, the scope of which is defined by the claims and their equivalents.
Reference to the literature
[1]Zhang Y，Mcneill E，Tian H，et al.Urine derived cells are a potential source for urological tissue reconstruction[J].J Urol，2008，180(5)：2226-2233.
[2] Shen Liangliang Ind, zhengling, twaing, et al, tissue origin of human urine derived stem cells and multipurpose research progress [ J ]. J. Of clinical urology, 2019, 34 (9): 745-748.
[3]Kang H，Choi S，Kim B，et al.Advanced properties of urine derived stem cells compared to adipose tissue derived stem cells in terms of cell proliferation，immune modulation and multi differentiation[J].J Korean Med Sci，2015，30(12)：1764-1776.
[4]Chun S，Kim H，Kwon S，et al.The efficacy and safety of Collagen-I and hypoxic conditions in urine-derived stem cell ex vivo culture[J].Tissue Eng Regen Med，2016，13(4)：403-415.
[5] Wu Qingjian, chen Wei, yang Fan, et al, research on the regulation of stem cell stem activity of long-chain non-coding RNA KCNQ1DN on urine-derived stem cells [ J ]. Journal of clinical urology, 2017, 32 (4): 290-293.
[6]Lu B，Atala A.Small molecules and small molecule drugs in regenerative medicine[J].Drug Discov Today，2014，19(6)：801-808.
[7]Deldar Y，Zarghami F，Pilehvar-Soltanahmadi Y，et al.Antioxidant effects of chrysin-loaded electrospun nanofibrous mats on proliferation and stemness preservation of human adipose-derived stem cells[J].Cell Tissue Bank，2017，18(4)：475-487.
[8]Chen A，Pi J，Hu J，et al.Identification and characterization of two morphologically distinct stem cell subpopulations from human urine samples[J].Sci China Life Sci，2020，63(5)：712-723.
[9]O′Brien P，Carrasco-Pozo C，Speisky H.Boldine and its antioxidant or health-promoting properties[J].Chem Biol Interact，2006，159(1)：1-17.
[10] Xie Zhenhua, wu Yaojiong. Mutual antagonism of expression levels of major dry genes and senescence-associated genes [ J ]. Biochemical and biophysical advances, 2014, 41 (7): 627-631.
Claims (4)
1. A method for enhancing the dryness of human urine derived stem cells, hscs, comprising the steps of:
(1) Isolating human urine-derived stem cells hscs from healthy adult sterile urine;
(2) The boldine is added into the basal culture medium of the hUSCs, so that the dryness of the hUSCs can be enhanced, and the hUSCs can be amplified and cultured.
2. A method of enhancing the dryness of human urine derived stem cells, hscs, according to claim 1, wherein: the step (1) is to obtain cell sediment by centrifuging sterile urine under normal temperature, inoculate and culture the cell sediment by using a basic culture medium of the hUSCs and screen the hUSCs, wherein the screened and cultured hUSCs have biological characteristics of mesenchymal stem cells, and the biological characteristics comprise cell wall-attached growth, high expression of CD44 and CD90 cell surface antigens.
3. A method of enhancing the dryness of human urine derived stem cells, hscs, according to claim 1, wherein: the effective final concentration of the Bohr alkali is 1.5-3 mu M, and the action time of the Bohr alkali on each generation of hUSCs is 0-72 h, namely, the Bohr alkali plays a role in enhancing the cell stem property in the whole course from the inoculation of the hUSCs in a pore plate to the cell expansion for 72h before secondary culture.
4. A method of enhancing the dryness of human urine derived stem cells, hscs, as claimed in claim 3, wherein: the Boerdines exert the effect of enhancing the cell stem property in the whole process and are used for enhancing the expression of stem genes OCT4 and MYC of hUSCs.
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| CN110499279A (en) * | 2019-01-25 | 2019-11-26 | 北京中医药大学 | A method of induce human urine derived stem cells to hepatocyte differentiation |
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| CN107254432A (en) * | 2017-08-16 | 2017-10-17 | 四川大学华西医院 | Culture medium for simultaneously separating two subsets of urine-derived stem cells, separation method and application |
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