CN106636210B - Transcription factor combines the method that induced fibroblast transdifferentiation is class interstitial glands - Google Patents
Transcription factor combines the method that induced fibroblast transdifferentiation is class interstitial glands Download PDFInfo
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Abstract
The present invention relates to a kind of methods that induced fibroblast transdifferentiation is class interstitial glands, it is characterised in that required transcription factor is cloned into slow virus carrier respectively, virion is packaged into and transfects to fibroblast；It is induction type interstitial glands using the method induced fibroblast transdifferentiation for being overexpressed transcription factor, wherein the transcription factor derives from people, mouse or rat, and the transcription factor is the combination of the transcription factor some or all of in the group being made of Dmrt1, Nr0b1, Sp1, Wt1, Nr4a1, Nr5a2, AP1, Creb1, Smad3, Nr5a1 and Gata4.The express spectra for the androgen synthesis related gene that cell after transdifferentiation has mature interstitial glands similar, and can synthesize and Testosterone Secretion.The class interstitial glands prepared through the invention can be used for treating male hypogonadism syndrome.
Description
Technical field
The invention belongs to fields of biomedicine, are related to process of cells transdifferentiate and application thereof, and in particular to utilize slow virus
It is the class interstitial tissue[of testis] that secreting function is synthesized with androgen that carrier, which expresses a variety of transcription factor induced fibroblast transdifferentiations,
The method of cell, while utilizing in this type Allografts of Leydig Cells to male interstitial tissue[of testis], to treat male gonad function
Decline syndrome provides new treatment method.
Background technique
Interstitial glands are the main sources of male testosterone, male sperm are occurred, Sex Differentiation and secondary
The development and maintenance of sign play a crucial role.Interstitial glands synthesize Ability of Testosterone after puberty with the age
Increase and is gradually reduced.At the same time, the factors such as environmental pollution, the accelerating rhythm of life and aging of population make between testis
The hypoandrogenism or deficiency disease that cell plastid itself dysfunction causes, the i.e. morbidity of hypogonadism (hypogonadism)
Rate increases year by year.Traditional male sex hormone carries out replacement therapy and has certain effect to hypogonadism, but this treatment is wanted
Last for life, side effect is very significant, is easy to appear the complication such as liver function impairment, Sperm specific enzyme reduction, prostate cancer, has
Patient is returned while side effect brings great economic pressures (Wang etc., J.Androl., 2009,32 (1): 1-10；Chen
Deng Endocrindogy, 2002,143 (5): 1637-1642).
With the development of tissue engineering technique, domestic and international expert attempts to have carried out close relative's donor graft art or stem cell transplantation
Art treats hypogonadism, achieves preferable effect.But interstitial glands exist only in testis, and limited amount (only accounts for testis
The 2-5% of ball cell total amount), although having there is more mature separation method to interstitial glands, in separation process
Loss cell it is inevitable, and grow up interstitial glands cannot carry out mitosis, immature interstitial glands again
Its splitting characteristic can be lost again in follow-up cultivation, these factors exacerbate the rare of interstitial tissue[of testis] seed cell.In addition, this
Transplanting generally by allogeneic supply, patient is postoperative to need to take for a long time immunosuppressor, while side effect is big also subject to
A series of ethics dispute, significantly limit the extensive use of the technology clinically.
Although mescenchymal stem cell or embryonic stem cell can also be used for being induced to differentiate between the class testis that can generate steroids
Cell plastid, but these cells are used for the defect that clinical research remains following several respects: (1) induce stem cell or
IPSCs (induced pluripotent stem cells, that is, the multipotential stem cell of induction) is divided into interstitial glands
Differentiation efficiency and purity are all very low, and the obtained steroidogenic ability of cell is very limited；(2) embryonic stem cell, which faces, exempts from
Epidemic disease is repelled, ethics morals problem and implant site form teratoma risk；(3) mescenchymal stem cell phenotype is inhomogenous, identification
Without clear standard, isolates and purifies and massive amplification faces technical difficulty；(4) although iPSCs has evaded embryonic stem cell bring
Ethics problem, but the reliability in its source and technical security are still the biggest obstacle faced in clinical application.
Adult cell transdifferentiation technology has induction time short compared with stem cell directional differentiation technique, and induced efficiency is high,
Cellular neoplastic is low, and transdifferentiation process is not need to rely on the apparent advantage such as division of cell, thus is considered as future
The important technological means of Homo sapiens reproduction's medicine.Body cell transdifferentiation can be directly other cell types by the technology, for regeneration
Medicine provides extremely valuable resource, it becomes the cell that the special any pedigree of patient is obtained with non-embryonic material can
Energy.Some researches show that transcription factor can be induced a plurality of types of cells of fibroblast transdifferentiation, including the heart in recent years
Myocyte, nerve cell, hepatic lineage, cartilage cell etc., these have all fully demonstrated transdifferentiation technology in regenerative medicine field
Tremendous potential.If by fibroblast transdifferentiation can be the class interstitial glands for having androgen synthesis and secreting function,
Allografts of Leydig Cells seed cell source deficiency and two realities of immunological rejection in clinical cytology treatment can be then solved simultaneously
Border problem.This is the focus of current this field research.But there is presently no about utilization which kind of transcription factor or transcription factor
Combination can induce the relevant report of class interstitial glands.
Summary of the invention
The shortcomings that in view of the prior art, is combined the invention mainly solves the technical problem of providing a kind of using transcription factor
Induced fibroblast transdifferentiation is the method for class interstitial glands.The class interstitial glands obtained by this method
(Induced Leydig-like cells, iLCs) can be used for treating male gonad function and lack syndrome.
In a first aspect, the present invention provide it is a kind of using transcription factor combination induced fibroblast transdifferentiation be class testis
The method of interstitial cell is packaged into virion the method includes required transcription factor is cloned into slow virus carrier respectively
And it transfects to fibroblast；Using the method induced fibroblast transdifferentiation of overexpression transcription factor between induction type testis
Cell plastid.
It should be appreciated by those skilled in the art that can extract and be selected from from animal for fibroblast of the invention
Embryo fibroblast, adult fibroblast break up institute by gene engineering method inducing embryo stem cell or iPSCs
The fibroblast or the fibroblast obtained extracts and is selected from from human body adult fibroblast or pass through gene
Engineering method inducing embryo stem cell or iPSCs break up resulting fibroblast (Yang Y, et al.Stem cells
and development,2015,24(4):459-70；Sonoyama T,et al.Endocrinology,2012,153(9):
4336-45).Available fibroblast can derive from skin, such as the fibroblast of rat-tail or application on human skin, can also be with
It is internal organ fibroblast (for example, fibroblast of heart etc.) either embryo fibroblast.
It can be extracted from corresponding animal or human body cell for transcription factor of the invention, source may include,
But it is not limited to, people, mouse and rat.And the nucleotide sequence of these transcription factors can be according to the side described in Itatura et al.
Method (Science, 1977,198:1056-1063) Lai Hecheng, also can use techniques known in the art from host genome
Obtain and identify gene.Techniques known in the art includes carrying out the synthesis of gene, the building of clone and expression vector, DNA sequence
The operation such as column analysis and identification, the conversion of host cell and culture and the separation of expression product identification is (referring to Sambrook et
al.,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory
Press,Cold Spring Harbor,NY,1989)。
For transcription factor of the invention be selected from Dmrt1, Nr0b1, Sp1, Wt1, Nr4a1, Nr5a2, AP1, Creb1,
Smad3, Nr5a1 or Gata4.Preferably, for transcription factor of the invention be selected from by Dmrt1, Nr0b1, Sp1, Wt1,
The combination of transcription factor some or all of in the group of Nr4a1, Nr5a2, AP1, Creb1, Smad3, Nr5a1 and Gata4 composition.
Transcription factor combination can for Dmrt1, Nr0b1, Sp1, Wt1, Nr4a1, Nr5a2, AP1, Creb1, Smad3, Nr5a1 and
The combination of Gata4；The combination of Dmrt1, Nr0b1, Sp1, Wt1, Nr4a1, Nr5a2, AP1, Nr5a1 and Gata4；Dmrt1,Sp1,
The combination of Wt1, AP1, Nr5a1 and Gata4；The combination of Dmrt1, Wt1, AP1, Nr5a1 and Gata4；Dmrt1,Sp1,Wt1,
The combination of Nr5a1 and Gata4；The combination of Dmrt1, Sp1, AP1, Nr5a1 and Gata4；Dmrt1, Nr5a1, Gata4 and Wt1's
Combination or the combination of Nr5a1, Gata4, Dmrt1 and AP1；Or the combination of Dmrt1, Gata4 and Nr5a1.
Optimal transcription factor group is combined into the combination of Dmrt1, Nr5a1 and Gata4；Followed by Dmrt1, Nr5a1, Gata4
The combination of combination or Nr5a1, Gata4, Dmrt1 and AP1 with Wt1；Be again Dmrt1, Sp1, Wt1, AP1, Nr5a1 and
Gata4；It is finally the combination of 11 kinds of factors, i.e. Nr0b1, Sp1, Wt1, Nr4a1, Nr5a2, AP1, Creb1, Smad3, Nr5a1
With the combination of Gata4.
Heretofore described transcription factor Dmrt1 (Doublesex and Mab-3Related Transcription
Factor 1) nucleotides sequence is classified as SEQ ID Nos:1-3；
Heretofore described transcription factor Nr0b1 (nuclear receptor subfamily 0group B
Member 1) nucleotides sequence is classified as SEQ ID Nos:4-6；
Heretofore described transcription factor Spl (transcription factor Sp1) nucleotides sequence is classified as SEQ ID
Nos:7-9；
Heretofore described transcription factor Wt1 (Wilms tumor 1) nucleotides sequence is classified as SEQ ID No:10-12s；
Heretofore described transcription factor Nr4a1 (nuclear receptor subfamily 4group A
Member 1) nucleotides sequence is classified as SEQ ID Nos:13-15；
Heretofore described transcription factor Nr5a2 (nuclear receptor subfamily 4group A
Member2) nucleotides sequence is classified as SEQ ID Nos:16-18；
Heretofore described transcription factor AP-1 1 (Jun proto-oncogene) nucleotides sequence is classified as SEQ ID Nos:
19-21；
Heretofore described transcription factor Creb1 (cAMP responsive element binding protein
1) nucleotides sequence is classified as SEQ ID Nos:22-24；
Heretofore described transcription factor Smad3 (SMAD family member 3) nucleotides sequence is classified as SEQ ID
Nos:25-27；
Heretofore described transcription factor Nr5a1 (nuclear receptor subfamily 5, group A,
Member 1) nucleotides sequence is classified as SEQ ID Nos:28-30；
Heretofore described transcription factor Gata4 (GATA binding protein 4) nucleotides sequence is classified as SEQ ID
Nos:31-33.
In preferred embodiments, induced fibroblast transdifferentiation is combined using transcription factor the present invention relates to a kind of
For the method for class interstitial glands, method includes the following steps:
1) it separates and purifies the fibroblast for viral vector infection；These fibroblasts can derive from skin
(rat-tail or application on human skin), internal organ fibroblast (heart etc.) or embryo fibroblast；
2) nucleotide sequence for 11 kinds of transcription factors that fibroblast transdifferentiation is class interstitial glands is obtained,
And it is cloned into Lentiviral pLVX-EF1 α-IRES-ZsGreen1 (Cat:#631982, Clonetch, Dalian treasured respectively
Bioengineering Co., Ltd, China), which can express green fluorescence as reporter gene；
3) by 11 kinds express transcription factors slow virus carrier transfect respectively 293T cell (Cat:#632180,
Clonetch, Dalian treasured bioengineering Co., Ltd, China) in, obtain the virion of integral packaging；
4) the mouse embryonic fibroblasts strain of bright cherry-red fluorescent reporter protein (cherry) is expressed in building；First with chain
Formula polymeric enzyme reaction (PCR) acquisition cholesterol side-chain cleavage gene cyp11a1 (cytochrome P450, family 11,
Subfamily a, polypeptide 1) promoter sequence and encode the fusion dna piece of bright cherry-red fluorescent reporter protein gene
Section (SEQ ID No:34), and it is cloned into slow virus carrier (pEZX-LvPM02 is purchased from GeneCopoeia Inc., USA)
In；After the virion for obtaining integral packaging, transfected fibroblast selects positive colony after screening 1 week using puromycin
And the fibroblast strain containing reporter gene is obtained with conventional cell culture processes；
5) by the lentiviral particle of 11 kinds of expression transcription factors of the Packing Intact 3) obtained, equivalent transfects 4) kind simultaneously and obtains
The fibroblast strain containing reporter gene obtained；It is positive thin using flow cytometry analysis expression red fluorescence after culture 3-7 days
The percentage of born of the same parents, and utilize I-125The content of testosterone in radioactivity enzyme-linked immunoassay method detection culture cell conditioned medium；
6) by the lentiviral particle of 11 kinds of expression transcription factors of the Packing Intact 3) obtained, 11 kinds of transcription factor groups are formed
It closes, contains 10 kinds of transcription factors in each combination respectively (i.e. every kind combination lacks a kind of different transcription factor)；By these turns
Combinations of factors is recorded, infects the fibroblast containing reporter gene according to the method described in 5), then red fluorescence is expressed in analysis
The percentage of positive cell when in cell conditioned medium testosterone content；These data and 5) the middle data obtained are compared and united
Meter analysis；It is class testis for then showing the transcription factor of missing there are the result of significant difference in fibroblast transdifferentiation
There is important regulating and controlling effect during interstitial cell；
7) 6) transcription factor that regulating and controlling effect is not significantly different in is excluded into (sharing 5 kinds), then by remaining transcription
The factor reconfigures (totally 6 kinds), then continues to screen according to method 6), final to obtain using testosterone yield as analysis indexes
Synthesis and the highest transcription factor combination of Testosterone Secretion amount；
8) according to the method described in 7), optimal transcription factor group is combined into Dmrt1, Nr5a1 and Gata4；Followed by
Dmrt1, Nr5a1, Gata4, Dmrt1 and Wt1 or Nr5a1, Gata4, Dmrt1 and AP1；Be again Nr5a1, Gata4,
Dmrt1, Sp1, Wt1 and AP1；Finally for 11 kinds of combinations of factors Nr0b1, Sp1, Wt1, Nr4a1, Nr5a2, AP1, Creb1,
Smad3, Nr5a1 and Gata4；
9) according to method 5), by optimal transcription factor combination transfection by 1) the isolated fibroblast of method, then
Culture 3-10 days, using molecular biology method, such as real-time fluorescence quantitative PCR, protein hybridization, genetic chip, detects these
Testosterone synthesizes and secretes the expression of each indispensable gene and albumen in access in transdifferentiated cells, and utilizes I-125Radioactivity enzyme
The yield of testosterone in linked immunoassay method detection culture cell conditioned medium；
10) cell after transdifferentiation is used to treat male gonad underactivity related disease.
It should be appreciated by those skilled in the art that can will be used to induced fibroblast transdifferentiation is class interstitial glands
Reagent be packaged into kit.Therefore, in second aspect of the present invention, the present invention also provides one kind to turn for induced fibroblast
Be divided into the kit of class interstitial tissue[of testis], the kit include for obtain fibroblastic reagent, slow virus carrier and
The combination of transcription factor.Wherein the transcription factor derives from people, mouse or rat, and the group of the transcription factor is combined into choosing
In the group of free Dmrt1, Nr0b1, Sp1, Wt1, Nr4a1, Nr5a2, AP1, Creb1, Smad3, Nr5a1 and Gata4 composition
The combination of part or all of transcription factor.For transcription factor it is combined select with above-mentioned induced fibroblast transdifferentiation for
The selection that the method for class interstitial tissue[of testis] combines transcription factor is identical.Fibroblast used be embryo fibroblast,
Internal organ fibroblast or skin fibroblasts.
In the third aspect, the present invention is provided by the class interstitial glands of the method induction differentiation of first aspect present invention,
The class interstitial glands can be used for treating male gonad function and lack syndrome.
In fourth aspect, the present invention is provided to be existed by the class interstitial glands of the method induction differentiation of first aspect present invention
Prepare the application in the kit for lacking syndrome for treating male gonad function.
At the 5th aspect, the present invention provides a kind of method treated male gonad function and lack syndrome, the method packet
It includes to inject into the interstitial tissue[of testis] of the two sides testis of patient with this need and a effective amount of be lured by the method for first aspect present invention
Lead the class interstitial glands of differentiation.
At the 6th aspect, the present invention provides a kind of kit for lacking syndrome for treating male gonad function, described
Kit includes by the class interstitial glands of the method induction differentiation of first aspect present invention.
Interstitial glands (Leydig cell, LC) are most important a kind of cells in mammal interstitial tissue[of testis], are male
Property body in androgen synthesis and secretion main source.Existing research shows that the Proliferation, Differentiation of interstitial glands is abnormal, counts
Amount is reduced and the decline of hormone sensitive lipase gene secreting function can all lead to internal hypoandrogenism, and then causes a variety of arrenotoky diseases
Occur.
The male gonad underactivity syndrome includes, but are not limited to caused by congenital hypogonadism, aging
Hypogonadism and interstitial glands damage and caused by hypogonadism.
The step of lacking syndrome using the class interstitial glands treatment male gonad function of transdifferentiation, is as follows:
1) the class interstitial glands of transdifferentiation are sorted out using flow cytometer and expands culture；
2) after will be cells trypsinised, phosphate buffer (PBS) washing and resuspension, adjustment density to every milliliter of 1-
5×106It is a；
3) it by after animal or people's anesthesia, is lain low in experimental bench, exposes two sides testis.Every side testis micro-injection
Device is by 106-107A cell infusion takes serum to detect Testosterone Content in Serum for 7-60 days after transplanting into interstitial tissue[of testis].
In conclusion the present invention provides following technical proposals:
1. a kind of method that induced fibroblast transdifferentiation is class interstitial glands, it is characterised in that by required transcription
Factor minute is not cloned into slow virus carrier, is packaged into virion and transfects to fibroblast；Utilize overexpression transcription factor
Method induced fibroblast transdifferentiation be induction type interstitial glands,
Wherein the transcription factor derive from people, mouse or rat, and the transcription factor be selected from by Dmrt1,
Some or all of in the group of Nr0b1, Sp1, Wt1, Nr4a1, Nr5a2, AP1, Creb1, Smad3, Nr5a1 and Gata4 composition
The combination of transcription factor.
2. the method according to the 1st, wherein the transcription factor group is combined into the combination of Dmrt1, Gata4 and Nr5a1.
3. the method according to the 1st, wherein the transcription factor group be combined into Dmrt1, Nr0b1, Sp1, Wt1,
The combination of Nr4a1, Nr5a2, AP1, Nr5a1 and Gata4.
4. the method according to the 1st, wherein the transcription factor group be combined into Dmrt1, Sp1, Wt1, AP1, Nr5a1 and
The combination of Gata4.
5. the method according to the 1st, wherein the transcription factor group be combined into Dmrt1, Wt1, AP1, Nr5a1 and
The combination of Gata4.
6. the method according to the 1st, wherein the transcription factor group be combined into Dmrt1, Sp1, Wt1, Nr5a1 and
The combination of Gata4.
7. the method according to the 1st, wherein the transcription factor group be combined into Dmrt1, Sp1, AP1, Nr5a1 and
The combination of Gata4.
8. the method according to the 1st, wherein the transcription factor group is combined into Dmrt1, Gata4, Nr5a1 and AP1
Combination or the combination of Dmrt1, Gata4, Nr5a1 and Wt1.
9. the method according to the 1st, wherein the transcription factor group be combined into Dmrt1, Nr0b1, Sp1, Wt1,
The combination of Nr4a1, Nr5a2, AP1, Creb1, Smad3, Nr5a1 and Gata4.
10. the method according to the 1st, wherein the fibroblast is embryo fibroblast, internal organ into fiber
Cell or skin fibroblasts.
11. it is a kind of for induced fibroblast transdifferentiation be class interstitial tissue[of testis] kit, the kit include use
In the combination for obtaining fibroblastic reagent, slow virus carrier and transcription factor, wherein the transcription factor is from people, small
Mouse or rat, and the group of the transcription factor be combined into selected from by Dmrt1, Nr0b1, Sp1, Wt1, Nr4a1, Nr5a2, AP1,
The combination of transcription factor some or all of in the group of Creb1, Smad3, Nr5a1 and Gata4 composition.
12. the kit according to the 11st, wherein the transcription factor group is combined into Dmrt1, Gata4 and Nr5a1
Combination.
13. the kit according to the 11st, wherein the transcription factor group be combined into Dmrt1, Nr0b1, Sp1, Wt1,
The combination of Nr4a1, Nr5a2, AP1, Nr5a1 and Gata4.
14. the kit according to the 11st, wherein the transcription factor group be combined into Dmrt1, Sp1, Wt1, AP1,
The combination of Nr5a1 and Gata4.
15. the kit according to the 11st, wherein the transcription factor group be combined into Dmrt1, Wt1, AP1, Nr5a1 and
The combination of Gata4.
16. the kit according to the 11st, wherein the transcription factor group be combined into Dmrt1, Sp1, Wt1, Nr5a1 and
The combination of Gata4.
17. the kit according to the 11st, wherein the transcription factor group be combined into Dmrt1, Sp1, AP1, Nr5a1 and
The combination of Gata4.
18. the kit according to the 11st, wherein the transcription factor group is combined into Dmrt1, Gata4 and Nr5a1
Combination.
19. the kit according to the 11st, wherein the transcription factor group be combined into Dmrt1, Nr0b1, Sp1, Wt1,
The combination of Nr4a1, Nr5a2, AP1, Creb1, Smad3, Nr5a1 and Gata4.
20. the kit according to the 11st, wherein fibroblast used in the kit is embryo fibroblast
Cell, internal organ fibroblast or skin fibroblasts.
21. a kind of method treated male gonad function and lack syndrome, the method includes to patient with this need
Two sides testis interstitial tissue[of testis] in injection any one of a effective amount of 1-10 the class interstitial tissue[of testis] that breaks up of method induction
Cell.
22. a kind of kit treated male gonad function and lack syndrome, the kit includes by 1-10
The class interstitial glands of the method induction differentiation of any one.
The more traditional stem cell method of inducing differentiation of the present invention has embodied two clear superiorities: 1) it is time-consuming short, at fiber
In cell the 3rd day after overexpression transcription factor, the expression of androgen synthesis key gene can be detected in host cell,
Its expression quantity and the ability for synthesizing testosterone are continuously increased with the extension of incubation time, by the 10th day after reach stable.Entire point
Change process is significantly better than the time required to traditional iPSCs induction differentiation (20 days or more)；2) risk and ethics of teratoma have been evaded
Problem.Since cell quantity does not have a had significant proliferation during transdifferentiation, and atomization is not through the multipotential stem cell stage, because
And the problems such as having evaded the risk and Medical Ethics of teratoma；3) donorcells can be from by employment itself.
Detailed description of the invention
From detailed description with reference to the accompanying drawing, features described above of the invention and advantage be will be apparent from, in which:
The agarose gel electrophoresis analysis of 11 kinds of Fig. 1, PCR amplification transcription factor genes.M, DNA molecular amount gradient；1,
Dmrt1；2, Nr0b1；3, Sp1；4, Wt1；5, Nr4a1；6, Nr5a2；7, AP1；8, Creb1；9, Smad3；10, Nr5a1；11,
Gata4。
The combination inducing embryo fibroblast transdifferentiation that Fig. 2,11 kinds of transcription factors form is class interstitial glands.
(A) cellular morphology after transdifferentiation；(B) cell synthesizes the content with Testosterone Secretion after transdifferentiation；Flow cytometer showed after (C, D) transdifferentiation
As a result.
The transcription factor combined sorting interpretation of result of Fig. 3, induced fibroblast transdifferentiation.(A, C, E) different transcription because
Flow cytometer showed result after sub-portfolio induced fibroblast transdifferentiation；Reporter gene is positive after (B, D, F, G, H) flow cytometer showed
Cell rate statistical results chart；(I) cell synthesis and the case where Testosterone Secretion after different transcription factors combination inductions；Data are with mean
± standard deviation (Mean ± SD) indicates that P < 0.05 indicates that P < 0.01 is indicated with * * with *, and P < 0.001 is indicated with * * *；N=4
Representing notebook data is obtained by 4 repetition analysis of experimental results.
Fig. 4, the androgen synthesis associated protein expression of class interstitial glands and testosterone synthesize variation tendency.(A) turn to divide
Change the immunofluorescence results of interstitial glands labelled protein in cell；(B) protein hybridization experimental result；(C) class interstitial tissue[of testis]
The analysis of cell synthesis testosterone yield.
The recovery of the low rat model serum testosterone of androgen is analyzed after Fig. 5, class Allografts of Leydig Cells.(A) thin
Born of the same parents' graft procedure process；(B) testis slice hematoxylin eosin staining analysis before and after cell transplantation；(C) 7 days serum after EDS modeling
Testosterone variation；(D) after cell transplantation 4,14,21,28 days model rat blood serum testosterone concentration variation；(E) class interstitial tissue[of testis] is thin
Rat body weight changes after born of the same parents' transplanting；(F) rat testicle weight change after cell transplantation；Data indicate with mean ± standard deviation, P <
0.05 is indicated with *, and P < 0.01 is indicated with * *, and P < 0.001 is indicated with * * *；Data do not have significant difference between ns representative group.
Fig. 6, the distribution after class Allografts of Leydig Cells 14 days and 28 days in rat testicle and interstitial glands are special
Foreign preteins expression analysis.
Initialism: GFP represents green fluorescent protein；
Merge represents two kinds of fluorescence coincidence patterns；
CYP11A1 represents cholesterol side-chain cleavage；
CYP17A1 represents 17 α-hydroxylase, 17,20- lyases；
StAR represents steroid hormone synthesized acute regulation protein；
HSD3B represents hydroxyl-δ -5- steroid dehydrogenase, 3 β-sterol δ isomerase；
HSD17b3 represents 17beta-Hydroxysteroid dehydrogenase 3；
LHCGR represents luteinizing principle receptor；
The ethane bismethane sulphur sulfone that EDS is represented；
Control represents control cell；
ILCs represents transdifferentiated cells；
MEFs represents fibroblast；
Testis represents animal testis；
Model representative model animal processing group
Sequence table explanation
For the sake of clarity, each sequence in rear attached sequence table is briefly described below:
The sequence number and its relevant information of the heretofore described transcription factor/fusion protein of table 1.
Specific embodiment
It is next by the following examples that the present invention is furture elucidated.However, it should be understood that the embodiment is merely illustrative
Purpose, be not intended to limit scope and spirit of the present invention.It should be noted that those skilled in the art should understand that
According to existing technical method, primer sequence needed for reagent used in of the invention and the following example, enzyme, PCR react etc.
It unless otherwise indicated, is the primer of the reagent that pure rank is analyzed commercially available from Reagent Company, enzyme and designed, designed.
Embodiment 1, the building of 11 kind of transcription factor Lentiviral and virus packaging
Firstly, using RNA extraction agent box, (Cat.#:74134, Qiagen are good for the limited public affairs of logical sequence biotechnology purchased from Guangzhou
Department, China) total serum IgE is obtained from mouse testis.Then PrimeScript is recycledTMII 1st Strand cDNA
Synthesis Kit (Cat.#:6210A, Dalian treasured Biotechnology Co., Ltd, China) synthesis cDNA, is placed in -80 DEG C of guarantors
It deposits.Separately designed by Huada gene company and synthesize for expand transcription factor Dmrt1, Nr0b1, Sp1, Wt1, Nr4a1,
The upstream and downstream primer of Nr5a2, AP1, Creb1, Smad3, Nr5a1 and Gata4 gene, and distinguish positive with reverse primer both ends
It introduces restriction enzyme site (underscore part, table 2).
According to conventional PCR method, using mouse testis cDNA as template, adds upstream and downstream primer (table 2), then set up
PCR reaction condition, circulation is entered after 94 DEG C of denaturation 3min, and loop parameter is 94 DEG C and is denaturalized 30 seconds that 55 DEG C are annealed 15 seconds, and 72 DEG C are prolonged
It stretches 30 seconds, totally 30 recycle.Electrophoresis detection target DNA fragment (Fig. 1).The restriction enzyme of standard is established according to existing method
Then enzymic digestion system (is purchased from New using BamH I (being purchased from New England Biolabs, Inc., USA) and EcoR I
England Biolabs, Inc., USA) it handles and recycles these target DNA fragments.Then, restriction enzyme identical as utilization
Enzymatic treatment and recycle Lentiviral pLVX-EF1 α-IRES-ZsGreen1 be connected, construct recombinant expression matter respectively
Grain pLVX-Dmrt1, pLVX-Nr0b1, pLVX-Sp1, pLVX-Wt1, pLVX-Nr4a1, pLVX-Nr5a2, pLVX-AP1,
PLVX-Creb1, pLVX-Smad3, pLVX-Nr5a1 and pLVX-Gata4 (concrete operations refer to Sambrook et al.,
Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory Press,
Cold Spring Harbor,NY,1989).The target DNA fragment of cyp11a1-mcherry will be encoded using identical method
(SEQ ID No:34) is with the Lentiviral pEZX-LvPM02 for after digestion with restriction enzyme, being connected to same treatment
In (being purchased from GeneCopoeia Inc., USA), recombinant expression plasmid pEZX-cyp11a1-mcherry is constructed.
It recovers and cultivates 293T cell (Cat:#632180 is purchased from Dalian treasured bioengineering Co., Ltd, China), then
By 1.5 × 106A 293T cell is layered on 10cm2Culture dish on, be added 10 milliliters of DMEM containing 10% fetal calf serum
(Dulbecco's Modified Eagle Medium) culture medium (Cat.#:12100-046, invitrogen Inc.,
USA), cultivated 48 hours in 37 DEG C of incubators containing 5% carbon dioxide.According to slow virus package kit (Cat.#:
631247, Lenti-XTMHTX Packaging System, Clontech Inc.USA) operating instruction progress slow virus packaging.
In short, in sterile PA tube, by the slow virus expression plasmid of 2.5 μ g and 5.0 μ l (0.5 μ g/ μ l) Lenti-Pac
HIV mixture (Cat.#:HPK-LvTR-20, GeneCopoeia Inc., USA) is diluted to the Opti-MEMI solution of 200 μ l
In (Cat.#:31985-062, invitrogen Inc., USA), incubation at room temperature formed compound after 25 minutes.By the compound
It is directly appended in the culture dish containing 293T cell, 37 DEG C of overnight incubations (12 hours).Replace fresh DMEM medium
(Cat.#:12100-046, invitrogen Inc., USA) and continues to put back in carbon dioxide incubator and cultivate 48-72 hours.
It is collected with 15 milliliters of centrifuge tubes and contains virulent culture medium, 500x g is centrifuged 10 minutes, is collected supernatant and is detected using PCR method
After the titre of slow virus, dispenses and be stored in spare in -80 DEG C.
Embodiment 2, for transdifferentiation embryo fibroblast and adult fibroblast separation
In order to obtain embryo fibroblast (mouse embryonic fibroblasts, MEFs), pregnancy is put to death first
Then -14.5 days 12.5 days pregnant mouse with scissors hara kiri and uterus, take out embryo.Embryo's week is rejected with aseptic nipper again
Four limbs, head and the internal organ of the coating, embryo that enclose.Embryonic tissue block is transferred in penicillin bottle with cutting after phosphate buffer cleaning
Knife rubs, and 2 milliliters of pancreatin are added, and 37 DEG C are incubated for 10 minutes.250xg is centrifuged 5 minutes, abandons supernatant, with containing 10% cow's serum and
Cell is resuspended in the DMEM culture medium (Cat.#:12100-046, invitrogen Inc., USA) of antibiotic, and is inoculated into culture
In ware；After culture 24 hours, fresh DMEM medium is replaced；It covers with, can harvest and is frozen in liquid nitrogen to cell.
Separation for adult fibroblast, the present invention use Mouse Tail-tip fibroblast (Tail-tip
Fibroblasts, TTFs).Firstly, clip adult mice tail point, phosphate buffer is rinsed.With syringe needle, by epidermis
After skin corium removing, remainder is twisted into 1cm with scissors3The tissue block of left and right.It is coated that tissue block is transferred to gelatin
In culture dish, and it is added in 6 milliliters of DMEM culture mediums and cultivates 5 days in culture dish.Abandon old culture medium, phosphate buffer washing two
After secondary, pancreatin digests 1 minute, and blows and beats into cell suspension.Cell suspension is collected into 15 milliliters of centrifuge tubes, 250xg centrifugation 5
Minute.Supernatant is abandoned, fresh culture culture is added to slow virus transformation experiment.
L cell strain (the cyp11a1- of embodiment 3, building expression reporter gene cyp11a1-mcherry
mcherry-Fibroblasts)
Sufficient amount of slow virus plasmid pEZX-cyp11a1-mcherry is obtained from embodiment 1, then by 1 × 108U
The slow virus of titre is added to containing 1 × 106In the culture dish of a MEFs or TTFs (separation of embodiment 2) and culture 24 is small
When.The DMEM culture medium (Cat.#:12100-046, invitrogen Inc., USA) containing virus is discarded, fresh DMEM training is replaced
Base is supported, after continuing culture 72 hours, the puromycin that final concentration of 2 mcg/ml (μ g/ml) is added is screened, continuous screen
It selects 1 week (Fig. 2A).Random picking positive colony is expanded, and is verified with molecular biology methods such as PCR and protein hybridizations.
Embodiment 4, transcription factor Dmrt1, Nr0b1, Sp1, Wt1, Nr4a1, Nr5a2, AP1, Creb1, Smad3, Nr5a1
It is interstitial glands with Gata4 combination inducing mouse embryonic fibroblast transdifferentiation
According in embodiment 3, by 1 × 106A fibroblast (cyp11a1-mcherry- containing reporter gene
Fibroblasts it) is inoculated into fresh DMEM medium.Culture 72 hours after, by 11 kinds of transcription factor lentiviral particles according to
Equal quality ratio mixing (total mass concentration of all virions is 10 mcg/mls), is then added in cell culture medium
Infection culture 24 hours.Remove the culture medium containing virus, replacement fresh DMEM medium continues culture 7 days.Pancreatin digestion, utilizes
Flow cytomery can express the positive cell ratio of bright cherry-red reporter gene, meanwhile, utilize radioactivity enzyme-linked immunoassay method
Detect content (Liu et al., the Journal of steriod biochemistry and of testosterone in Transfected cells supernatant
molecular biology,2014,144:483-91)。
The experimental results showed that in the fibroblast (cyp11a1-mCherry-fibroblasts) containing reporter gene
After all 11 transcription factor infection are added, the positive cell ratio for expressing bright cherry-red reporter gene is 53% (Fig. 2 C, D), and
These positive cells have the ability of synthesis and Testosterone Secretion, and mean concentration reaches 8.46 nanograms/milliliters (ng/ml) (figure
2B).This result also prompts, and the combination of this 11 transcription factors composition can be tool synthesis point with induced fibroblast transdifferentiation
Secrete the class interstitial glands of Ability of Testosterone.
Embodiment 5, the screening for promoting the optimal transcription factor that fibroblast transdifferentiation is class interstitial glands to combine
By exclusive method one by one, the percentage for expressing bright cherry-red reporter gene positive cell will not significantly affected or testosterone closes
It is excluded at the transcription factor of yield.11 kinds of transcription factors are reconfigured, in each combination respectively containing 10 kinds transcription because
Sub (i.e. every kind combination lacks a kind of different transcription factor), then transfects the fibroblast strain containing reporter gene.Culture
After 3-10 days, the ratio of bright cherry-red reporter gene positive cell is expressed using flow cytometry analysis and is exempted from by the way that radioactivity is enzyme-linked
The content of epidemic disease method detection culture medium supernatant testosterone.
First round the selection result shows that removing Smad3 and Creb1 in the combination containing 11 kinds of transcription factors does not influence table
Up to the ability of ratio and cell the synthesis testosterone of bright cherry-red reporter gene positive cell, therefore, using Smad3 and Creb1 as non-
The required factor and exclude (Fig. 3 A, B).
Second wheel the selection result shows that deleting Nr5a2, Nr4a1 and Nr0b1 in 9 factors will not significant impact table
Up to the ability of the percentage when cell synthesis testosterone of bright cherry-red reporter gene positive cell, therefore, by Nr5a2, Nr4a1 and
Nr0b1 is also excluded from 9 factors as nonessential transcription factor, and remaining 6 factors are reserved for next round screening (figure
3C, D).
Third round the selection result is shown, SP1, AP1 or Wt1 are deleted in 6 factors and is equally not significantly affected by bright cherry-red report
The ability of the percentage when cell synthesis testosterone of gene masculine cell is accused, and deleting Dmrt1, Gata4 or Nr5a1 respectively can be significant
Influence to express bright cherry-red reporter gene positive cell average percent, make its from 49% be down to respectively 27.7%, 23.2% and
17.6%.And the percentage of bright cherry-red reporter gene cell is expressed after Dmrt1, Gata4 and Nr5a1 co-infection fibroblast
It is average then be 40.8% (Fig. 3 E)；And testosterone mean concentration reaches 23.4 and receives in the cells and supernatant after the combination transdifferentiation
Grams per milliliter (ng/ml) has raised 3.1 times and 2.76 times compared with 6 factors and 11 factors respectively.
It can be seen that Dmrt1, Gata4 and Nr5a1 group be combined into fibroblast transdifferentiation be class testosterone interstitial cell most
Excellent transcription factor combines (Fig. 3 E-G).AP1 or Wt1 is added in this 3 factor will not dramatically increase bright cherry-red reporter gene sun
Property cell percentage, but can improve transdifferentiated cells synthesis testosterone ability (Fig. 3 G, H, I).Therefore, Dmrt1, Gata4,
The combination of Nr5a1 and AP1 or the combination of Dmrt1, Gata4, Nr5a1 and Wt1 are also to have to remarkably promote fibroblast turn point
Turn to the ability of the class interstitial glands of tool testosterone synthesis capability.
Embodiment 6, class interstitial glands expression interstitial glands specific marker proteins and testosterone synthesis capability point
Analysis
Whether the cell after immunofluorescence, Western identification transdifferentiation expresses Leydig cell key albumen, as a result shows
Show, cell after transdifferentiation expression cholesterol side-chain cleavage (cytochrome P450, family 11, subfamily a,
Polypeptide 1, Cyp11a1), steroid hormone synthesized acute regulation protein (Steroidogenic acute
Regulatory protein, StAR), hydroxyl-δ -5- steroid dehydrogenase, 3 β sterol δ isomerase, 1 (hydroxy-delta-
5-steroid dehydrogenase, 3beta-and steroid delta-isomerase 1, hsd3b1), 17 beta-hydroxies
Steroid dehydrogenase 3 (hydroxysteroid (17-beta) dehydrogenase 3, hsd17b3) and luteinizing principle
Receptor protein (Luteinizing hormone receptor, LHCGR) (Fig. 4 A), protein hybridization experiment further demonstrate this
A little results (Fig. 4 B).The testosterone synthesis capability of fibroblast and transdifferentiated cells is analyzed, the results show that transfection 2 days
Afterwards, it can be detected testosterone in the culture supernatant of transdifferentiated cells, and testosterone concentration gradually rises with the extension of incubation time
Height reaches peak (Fig. 4 C) on the 10th day.And as control, fibroblast can neither express interstitial glands specific protein
It is white, do not have synthesis Function of Testosterone (Fig. 4 C) yet.
Embodiment 7 establishes adult interstitial glands " knockout " mouse model
Adult rat or mouse testis contain a large amount of adult interstitial glands, these cells largely can synthesize and divide
Secrete male sex hormone.According to 90 mg kg of body weights of dosage, male mouse (be purchased from Guangdong Province's medical animal experiment center, 12 weeks
Age) intraperitoneal injection ethane bismethane sulphur sulfone (Ethane Dimefhane sulfonaft, EDS).After injection 7 days, in mouse testis
Apoptosis can occur for all maturation interstitial glands (ALCs), and Testosterone Content in Serum will settle to the same position of radioactivity in animal body
The lower limit of plain enzyme-linked immune detection method.This adult interstitial glands " knockout " animal may be used as studying congenital sexual gland
Hypogonadism caused by hypofunction, aging and interstitial glands damage and caused by the diseases such as hypogonadism
Model.
Embodiment 8, transplanting class interstitial glands can promote the serum testosterone of interstitial glands " knockout " mouse to normal
It is horizontal
The class interstitial glands of transdifferentiation are used to restore in animal body the reality of the low model mouse serum testosterone of androgen
It is as shown in Figure 5A to test process.In short, carrying out modeling in male rat intraperitoneal injection EDS first, cell shifting is carried out after modeling 7 days
It plants.The class interstitial glands of transdifferentiation are washed into resuspension with phosphate buffer, adjustment cell density is every milliliter 1 × 107It is a.
After model mouse is anaesthetized with yellow Jackets, is lain low in experimental bench, expose two sides testis.Testis is wiped with 75% alcoholic solution
Then ball outer skin takes 1,000,000 class interstitial glands to be injected into interstitial tissue[of testis] with micro syringe.Let the acupuncture needle remain at a certain point after injection 5
Minute, it is further continued for injection other side testis.Difference 0 day after injection, 14 days, 21 days, serum and tissue sample is taken within 28 days to carry out
Detection.
Experimental result shows that 7 days after cell transplantation, model group interstitial glands are sharply reduced, and in convoluted seminiferous tubule
Cell illustrates that EDS optionally promotes interstitial glands that apoptosis occurs without significant changes.And the fibroblast transplanted
(MEFs) and class interstitial glands (iLCs) can be uniformly distributed (Fig. 5 B) in model mouse interstitial tissue[of testis].Serum testosterone detection knot
Fruit shows after transplanting that the serum testosterone of model group animal is remarkably decreased (Fig. 5 C) 7 days.And class Allografts of Leydig Cells 14 days
Afterwards, testing result is shown, the serum testosterone of model mouse is begun to ramp up, and 21 days whens restore to the 50% of normal level, and after 28 days
Normal level is substantially returned to, and the level of serum testosterone of fibroblast transplantation group and model group has no and is obviously improved (figure
5D)。
Since testosterone has important influence to male Body Weight and testicualr development, to rat body weight before and after cell transplantation
And testis weighing detection discovery, serum testosterone lowly can significantly cause the weight of rat and testicular weight to mitigate.Class interstitial tissue[of testis]
Cell transplantation can promote weight and testicular weight restores to normal level, and model group and Allogenic Cultured Dermal Fibroblasts Transplantation group are then without obvious
Help (Fig. 5 E, F).
The cellular localization after transplanting is tracked using green fluorescent reporter albumen, as a result, it has been found that, 14 days, 28 after transplanting
The class interstitial glands of its GFP label are distributed in interstitial tissue[of testis], while also expressing interstitial glands specific mark egg
White cholesterol side-chain cleavage (cytochrome P450, family 11, subfamily a, polypeptide 1,
Cyp11a1).Although the fibroblast (MEFs) of fluorescent protein labeling can also colonize in interstitial tissue[of testis], can not table
Up to interstitial glands Specific Marker Proteins (Fig. 6).
In conclusion can be colonized in male mouse interstitial tissue[of testis] after the class Allografts of Leydig Cells of transdifferentiation, while table
Up to interstitial glands Specific Marker Proteins, it can also synthesize and secrete androgen, these tool androgen synthesis functions turn to divide
Changing cell can promote the recovery of the low rat model serum testosterone of androgen, and has and treat congenital hypogonadism, declines
Hypogonadism caused by old and interstitial glands damage and caused by the diseases such as hypogonadism potential application valence
Value.
It should be understood that although carrying out particularly shown and description to the present invention with reference to its illustrative embodiment,
It should be understood by those skilled in the art that without departing substantially from by spirit and model of the invention as defined in the claims
Under conditions of enclosing, any combination of various embodiments can be carried out in the variation for wherein carrying out various forms and details.
Therefore, the scope of the present invention includes being equal to the modification used in purpose and range in claims.
Claims (2)
1. a kind of method that induced fibroblast transdifferentiation is class interstitial glands, it is characterised in that by required transcription factor
It is cloned into slow virus carrier respectively, be packaged into virion and transfects to fibroblast；Utilize the side for being overexpressed transcription factor
Method induced fibroblast transdifferentiation be induction type interstitial glands, wherein the fibroblast extracted from animal and
Selected from embryo fibroblast, adult fibroblast or pass through gene engineering method inducing embryo stem cell or iPSCs points
Change resulting fibroblast or the fibroblast extracts and is selected from from human body adult fibroblast or passes through
Gene engineering method inducing embryo stem cell or iPSCs break up resulting fibroblast,
Wherein the required transcription factor derives from people, mouse or rat, and the required transcription factor is transcription factor
Combination, the combination of the transcription factor are as follows:
The combination of Dmrt1, Gata4 and Nr5a1；
The combination of Dmrt1, Nr0b1, Sp1, Wt1, Nr4a1, Nr5a2, AP1, Nr5a1 and Gata4；
The combination of Dmrt1, Sp1, Wt1, AP1, Nr5a1 and Gata4；
The combination of Dmrt1, Wt1, AP1, Nr5a1 and Gata4；
The combination of Dmrt1, Sp1, Wt1, Nr5a1 and Gata4；
The combination of Dmrt1, Sp1, AP1, Nr5a1 and Gata4；
The combination of Dmrt1, Gata4, Nr5a1 and AP1；
The combination of Dmrt1, Gata4, Nr5a1 and Wt1；Or
The combination of Dmrt1, Nr0b1, Sp1, Wt1, Nr4a1, Nr5a2, AP1, Creb1, Smad3, Nr5a1 and Gata4.
2. according to the method described in claim 1, wherein the fibroblast is embryo fibroblast, internal organ into fiber finer
Born of the same parents or skin fibroblasts.
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