CN105062978A - Method for inducing umbilical cord mesenchymal stem cells to be differentiated into leydig cells through gold nanoparticle medicated gene transfection - Google Patents
Method for inducing umbilical cord mesenchymal stem cells to be differentiated into leydig cells through gold nanoparticle medicated gene transfection Download PDFInfo
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- CN105062978A CN105062978A CN201510498088.9A CN201510498088A CN105062978A CN 105062978 A CN105062978 A CN 105062978A CN 201510498088 A CN201510498088 A CN 201510498088A CN 105062978 A CN105062978 A CN 105062978A
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Abstract
The invention provides a method for inducing umbilical cord mesenchymal stem cells to be differentiated into leydig cells through gold nanoparticle medicated gene transfection. Plasmids with SF-1 (seroidogenic factor 1) genes and LHR (luteotropic hormone receptor) genes are cotransfected into the umbilical cord mesenchymal stem cells through gold nanoparticles, then the umbilical cord mesenchymal stem cells are induced by compounds or growth factors to be directionally differentiated into the leydig cells. The leydig cell differentiation method is efficient and safe, the differentiated leydig cells can serve as a research substitution model and can be used as seed cells of clinical cell treatment of the leydig cells, and an application prospect of leydig cell regeneration replacement treatment is widened.
Description
Technical field
The invention belongs to cytobiology and developmental biology field, be specifically related to a kind of method by nm gold particles mediated gene transfection induction human umbilical cord mesenchymal stem cells differentiation interstitial glands.
Background technology
Male climacteric syndrome is the atrophy due to testis, and the secretion of testosterone reduces, and the testis of atrophy reduces the reaction of gonad-stimulating hormone, makes the regulatory function of sexual hormoue in body unbalance and the series of symptoms that causes.Main manifestations is psychic symptoms: energy is not concentrated, hypomnesis, depressed, anxiety, and irritability is suspicious, neurotic, and work capacity declines.Vasomotor symptoms had palpitation, hectic fever, perspires.Property aspect the sexual interest of symptom reduce, sexual desire reduces, impotence.Physiology physical efficiency symptom has sleep to reduce, easily tired, poor appetite, bone and arthralgia.
The male hormone of current clinical main applied chemistry synthesis carries out replacement therapy.But human body prolonged application chemosynthesis male sex hormone easily causes the complication such as hepatic disorder, hyperlipidemia and prostate cancer, excessive the supplementing of short-term also can cause the multiple side reactions such as people's emotional lability.Close relative's donor graft art has been carried out in recent domestic expert trial or stem cell transplantation art treatment testis loses or the low disease of male gonad, obtains better effects.But there is the shortcomings such as donor source is limited, operation is complicated, funds are high in this transplantation, receptor needs long-term taking immunosuppressor etc.
Recently, the androgenic factor of interstitial glands synthesis secretion is regulated also more and more to be subject to people's attention.Through investigation, there is not yet the method relevant report through nm gold particles mediated gene transfection induction human umbilical cord mesenchymal stem cells differentiation interstitial glands.
Summary of the invention
An object of the present invention is to build a kind of method by nm gold particles mediated gene transfection induction human umbilical cord mesenchymal stem cells differentiation interstitial glands, mainly pass through the nanometer colloid gold grain of 10-30nm diameter by the Steroidgenesis factor 1 (SteroidogenicFactor1, and prolan B acceptor (luteotropichormonereceptor SF-1), LHR) plasmid co-transfection enters human umbilical cord mesenchymal stem cells, then be interstitial glands by compound or growth factor-induced human umbilical cord mesenchymal stem cells directed differentiation, and then secretion generates testosterone.
By a method for nm gold particles mediated gene transfection induction human umbilical cord mesenchymal stem cells differentiation interstitial glands, comprise following steps:
1) synthesis of nm gold particles, nm gold particles to the bag quilt of genophore, nm gold particles mediation gene transfection;
2) compound or growth factor-induced human umbilical cord mesenchymal stem cells directed differentiation is adopted to be interstitial glands
A described nanometer gold material is the nanometer colloid gold grain of diameter 10-30nm, preferred 20nm.
The building-up process of a described nanometer gold material is as follows:
(1) by hydrochloro-auric acid (HAuC1
4) be mixed with 0.01% aqueous solution, get 100mL and be heated to boil.
(2) 1% trisodium citrate (Na3C6H5O72H2O) aqueous solution of 1.5mL is accurately added under stirring.
(3) heated and boiled 15min is continued.Now can be observed flaxen aqueous solution of chloraurate to gray very soon after Trisodium Citrate adds, continue and change into black, being stable into redness gradually subsequently.Whole process is about 2-3min.
(4) original volume is returned to distilled water after being cooled to room temperature for subsequent use.
The gene bag quilt of described nm gold particles mediation and gene transfection, its step is as follows:
Human umbilical cord mesenchymal stem cells is carried out conventional adherent culture, by hole 4 × 10 every in 6 well culture plates
5individual cell, and by following amount of reagent operation: get two kinds and treat that Pignus pignoris grain (respectively containing Steroidgenesis Factor 1 Gene and prolan B acceptor gene) each 2 μ g are dissolved in 100 μ lPBS damping fluids, add 16 μ l nm gold particles solution again, vortex 1s mixes, and room temperature adds in cell after placing 2-5min; Transfection changes induction broth into after within 24 hours, changing nutrient solution;
Adopt compound or growth factor-induced human umbilical cord mesenchymal stem cells directed differentiation to be interstitial glands, add the inducing culture of following formula after 24 hours in transfection: 5 × 10
-7mol/L tretinoin and 1 × 10
-3the bromo-cAMP of mol/L8-, after differentiation-inducing 7 days, detects steroid synthesis associated protein in interstitial glands.
Beneficial effect:
The invention provides a kind of method being divided into interstitial glands by nm gold particles mediated gene transfection induction human umbilical cord mesenchymal stem cells, described method is efficient, safety, and the interstitial glands after differentiation can be used as research alternative model; Can be used for the seed cell of interstitial glands cell therapy clinically, improve the application prospect of interstitial glands regeneration replacement therapy.
Described method has opened up a kind of new stem cell gene transferring pathway, for differentiation of stem cells for interstitial glands provides a kind of efficient, safe method;
Interstitial glands after described method differentiation can as the alternative model of research interstitial glands regulation of secretion function effect Elements research.
Embodiment
Below by specific embodiment, technical scheme of the present invention is described in detail, but scope of the present invention is not by the restriction of these embodiments.
By a method for nm gold particles mediated gene transfection induction human umbilical cord mesenchymal stem cells differentiation interstitial glands, comprise following steps:
3) synthesis of nm gold particles, nm gold particles to the bag quilt of genophore, nm gold particles mediation gene transfection;
4) compound or growth factor-induced human umbilical cord mesenchymal stem cells directed differentiation is adopted to be interstitial glands
The building-up process of a described nanometer gold material is as follows:
(1) by hydrochloro-auric acid (HAuC1
4) be mixed with 0.01% aqueous solution, get 100mL and be heated to boil.
(2) 1% trisodium citrate (Na3C6H5O72H2O) aqueous solution of 1.5mL is accurately added under stirring.
(3) heated and boiled 15min is continued.Now can be observed flaxen aqueous solution of chloraurate to gray very soon after Trisodium Citrate adds, continue and change into black, being stable into redness gradually subsequently.Whole process is about 2-3min.
(4) original volume is returned to distilled water after being cooled to room temperature for subsequent use.
The gene bag quilt of described nm gold particles mediation and gene transfection, its step is as follows:
Human umbilical cord mesenchymal stem cells is carried out conventional adherent culture, by hole 4 × 10 every in 6 well culture plates
5individual cell, L-DMEM+5%FBS substratum, incubator temperature 37 degree, CO2 concentration is 5%, and by following amount of reagent operation: get two kinds and treat that Pignus pignoris grain (respectively containing Steroidgenesis Factor 1 Gene and prolan B acceptor gene) each 2 μ g are dissolved in 100 μ lPBS damping fluids, add 16 μ l nm gold particles solution again, vortex 1s mixes, and room temperature adds in cell after placing 2-5min; Transfection changes induction broth into after within 24 hours, changing nutrient solution;
Adopt compound or growth factor-induced human umbilical cord mesenchymal stem cells directed differentiation to be interstitial glands, add the inducing culture of following formula after 24 hours in transfection: 5 × 10
-7mol/L tretinoin and 1 × 10
-3the bromo-cAMP of mol/L8-, after differentiation-inducing 7 days, detects steroid synthesis associated protein in interstitial glands.
Radioimmunoassays is used to detect the situation of the interstitial glands Testosterone Secretion become by mescenchymal stem cell directed differentiation.
Concrete detection method is: testosterone secretion measures and carries out according to testosterone mensuration test kit specification sheets.Take out enzyme plate, add the standard substance of 50 μ L respectively in blank micropore according to order correspondence; Mark sample number into spectrum respectively, add 50 μ L samples in blank micropore; The enzyme labelling solution of 100 μ L is added in standard sample wells and sample well; 37 DEG C of incubation reaction 60min; Concentrated washing lotion and distilled water 1:20 doubly dilute rear cleaning of enzyme target 5 times, leave standstill 10-20S at every turn; Every hole adds substrate A, each 50 μ L of B liquid; Lucifuge incubation reaction 15min at 37 DEG C; Every hole adds 50 μ L stop buffers, termination reaction.The microplate reader of wavelength 450nm measures OD value, draws testosterone concentration value by typical curve.
Result shows, records solvent control group testosterone concentration 0.20nmol/L, inducing component and the cell that comes has the ability of Testosterone Secretion, and three times parallel laboratory test testosterone concentration is respectively
0.95nmol/L, 0.91nmol/L and 0.98nmol/L (see table 1), control group gained testosterone concentration is the serum background influence in differentiation agents.
Table 1: the content not breaking up and break up testosterone in rear cell culture fluid
| Group | Testosterone concentration (nM/L) |
| Control group | 0.20 |
| Parallel differentiation experiment 1 | 0.95 |
| Parallel differentiation experiment 2 | 0.91 |
| Parallel differentiation experiment 3 | 0.98 |
In the above-described embodiments, preferred forms of the present invention is described, obviously, under inventive concept of the present invention, still can make a lot of change.At this, should illustrate, any change made under inventive concept of the present invention all will fall within the scope of protection of the present invention.
Claims (6)
1., by a method for nm gold particles mediated gene transfection induction human umbilical cord mesenchymal stem cells differentiation interstitial glands, it is characterized in that comprising the following steps:
The synthesis of nm gold particles, nm gold particles to the bag quilt of genophore, nm gold particles mediation gene transfection;
Compound or growth factor-induced human umbilical cord mesenchymal stem cells directed differentiation is adopted to be interstitial glands.
2. the method for claim 1, is characterized in that: a described nanometer gold material is the nanometer colloid gold grain of diameter 10-30nm.
3. method as claimed in claim 1 or 2, is characterized in that: a described nanometer gold material is the nanometer colloid gold grain of diameter 20nm.
4. the method for claim 1, other are: the building-up process of described nm gold particles is:
(1) hydrochloro-auric acid (HAuC14) is mixed with 0.01% aqueous solution, gets 100mL and be heated to boil;
(2) 1% trisodium citrate (Na3C6H5O72H2O) aqueous solution of 1.5mL is accurately added under stirring;
(3) continue heated and boiled 15min, now can be observed flaxen aqueous solution of chloraurate and gray very soon after Trisodium Citrate adds, continue and change into black, being stable into redness gradually subsequently, whole process 2-3min;
(4) original volume is returned to distilled water after being cooled to room temperature for subsequent use.
5. method according to claim 1, is characterized in that: described nm gold particles is to the bag quilt of genophore, and the gene transfection process of nm gold particles mediation is:
Human umbilical cord mesenchymal stem cells is carried out conventional adherent culture, by hole 4 × 10 every in 6 well culture plates
5individual cell, and by following amount of reagent operation: get two kinds and treat that Pignus pignoris grain (respectively containing Steroidgenesis Factor 1 Gene and prolan B acceptor gene) each 2 μ g are dissolved in 100 μ lPBS damping fluids, add 16 μ l nm gold particles solution again, vortex 1s mixes, and room temperature adds in cell after placing 2-5min; Transfection changes induction broth into after within 24 hours, changing nutrient solution.
6. the method described in claim 1 or 5, is characterized in that: described employing compound or growth factor-induced human umbilical cord mesenchymal stem cells directed differentiation are interstitial glands, and inducing culture is: in basic medium, add 5 × 10
-7mol/L tretinoin and 1 × 10
-3the bromo-cAMP of mol/L8-.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105807050A (en) * | 2016-03-15 | 2016-07-27 | 威尚生物技术（合肥）有限公司 | Mumps virus antibody double-antigen sandwich method immune colloidal gold test strip and preparation method thereof |
| CN110423783A (en) * | 2019-07-10 | 2019-11-08 | 华中科技大学 | Gene transfection/drug targeting/signal transduction method and system based on negative magnetophoresis |
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| CN101892190A (en) * | 2010-06-24 | 2010-11-24 | 浙江大学 | Construction and application of model for differentiating stem cells into testicular interstitial cells |
| CN102174468A (en) * | 2011-02-24 | 2011-09-07 | 暨南大学 | Method and application for inducing human umbilical cord mesenchyme stem cells to be differentiated into testicular interstitial cells |
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| CN101892190A (en) * | 2010-06-24 | 2010-11-24 | 浙江大学 | Construction and application of model for differentiating stem cells into testicular interstitial cells |
| CN102174468A (en) * | 2011-02-24 | 2011-09-07 | 暨南大学 | Method and application for inducing human umbilical cord mesenchyme stem cells to be differentiated into testicular interstitial cells |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105807050A (en) * | 2016-03-15 | 2016-07-27 | 威尚生物技术（合肥）有限公司 | Mumps virus antibody double-antigen sandwich method immune colloidal gold test strip and preparation method thereof |
| CN110423783A (en) * | 2019-07-10 | 2019-11-08 | 华中科技大学 | Gene transfection/drug targeting/signal transduction method and system based on negative magnetophoresis |
| CN110423783B (en) * | 2019-07-10 | 2021-08-03 | 华中科技大学 | Gene transfection/drug targeting/signal conduction method and system based on negative magnetophoresis |
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Application publication date: 20151118 |