CN102417893A - Method for separating and culturing swine spermatogonial stem cells by using one-step enzyme process - Google Patents
Method for separating and culturing swine spermatogonial stem cells by using one-step enzyme process Download PDFInfo
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- CN102417893A CN102417893A CN2011102729277A CN201110272927A CN102417893A CN 102417893 A CN102417893 A CN 102417893A CN 2011102729277 A CN2011102729277 A CN 2011102729277A CN 201110272927 A CN201110272927 A CN 201110272927A CN 102417893 A CN102417893 A CN 102417893A
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Abstract
The invention discloses a method for separating and culturing swine spermatogonial stem cells by using the one-step enzyme process, and discloses a culture system. According to the invention, the one-step enzyme process is utilized to substitute the two-step enzyme digestion process frequently used at present for separation and digestion of swine spermatogonial stem cells, and has the advantages of easily available cells, short culture time, fast cell proliferation and a good stage of cells. The invention enables the existing problems of difficult separation of spermatogonial stem cells, small quantity, slow propagation and high requirements for culture to be overcome, and provides a novel method for rapid acquisition of considerable swine spermatogonial stem cells; and the method has the advantages of simple and convenient operation, low experiment cost, reduced risk of failed experiments and easy popularization and application.
Description
Technical field
The present invention relates to cellular segregation and cultivate the field, be specifically related to a kind of method of a step enzyme method separation and Culture pig stem spermatogonium.
Background technology
(Spermatogonial stem cells is uniquely in the male adult of mammal can genetic information be passed to follow-on stem cell SSCs) to stem spermatogonium, and it has multipotency; Can be induced to be divided into polytype cell, it can also generate embryoid body under a stable condition, is a kind ofly naturally just can obtain IPS cell good material without transgenic; And it generates sperm; Accomplish the transmission of gene, be accompanied by the rise of transgenic technology, it becomes a kind of new transgenic approach.
Spermatogeny is the process of a complicacy, is the prerequisite of keeping arrenotoky ability and life continuation.Understand spermatogenetic molecule and celelular mechanism in depth, the human endogenous SSCs that causes because of chemotherapy and radiation of protection Endangered animal species and treatment damages and the recovery male fecundity.The stem spermatogonium technology combines with animal seed selection work, will produce good domestic animal on a large scale.
SSCs has multipotency and is divided into and derives from tridermic multiple histocyte, and stem spermatogonium becomes possibility for the source of cell replacement therapy and histoorgan transplanting from now on.Its utilization in fields such as organizational project, Transplanted cells, gene therapies provides the research basis, and clinical medicine is had big using value.
Stem spermatogonium not only has special value to the research of aspects such as biology, biology techniques, medical science and auxology, makes it become domestic and international research focus in recent years.
The separation method of the main SSCs of research mainly concentrates on mechanical process and the digestion of two step enzyme methods at present; And purification process mainly contains: the differential adherent method, and the percoll density gradient centrifugation, the natural gravity settling process, methods such as sorting such as flow cytometry sorting and immunomagnetic beads, the whole bag of tricks all exists relative merits, selects as required.
Summary of the invention
The deficiency that exists in the objective of the invention is to cultivate according to existing cellular segregation provides a kind of acquisition cell easy, and incubation time is short, and cell proliferation is fast, state good and easy and simple to handle, the method for the separation and Culture pig stem spermatogonium that experimental cost is low.
Above-mentioned purpose of the present invention is achieved through following technical scheme:
A kind of method of a step enzyme method separation and Culture pig stem spermatogonium comprises the steps:
(1) testis of piglet is put into the container that fills PBS,, remove blood stains with PBS washing 2 ~ 3 times;
(2) remove albuginea testis and reticular tissue, remaining testis tissue is cut into small pieces, transfer in the petridish;
(3) add the PBS of 10 times of volumes,, leave standstill with suction pipe piping and druming, treat that testis tissue sinks to the bottom after, abandon supernatant;
(4) repeating step is (3) 2 ~ 3 times;
(5) collagenase digesting of 10 times of volumes of adding is in 37 ℃ water-baths, 100 rpm, then with suction pipe piping and druming, up to can't see tissue block;
(6) PBS of adding 5 ~ 10 ml, piping and druming at 16 ℃, centrifugal 5 min of 600 rpm, is abandoned supernatant, collects the bottom cell;
(7) with the outstanding cell of difference adherent culture basic weight, the adjustment cell concn is 5 * 10
6Individual/ml, be inoculated in the culturing bottle that prior usefulness 0.1 % gelatin encapsulates, in the sterile culture case, cultivate.
As a kind of preferred version, in the aforesaid method, the culture condition of said cell culture incubator is 37 ℃, 5 % CO
2, saturated humidity.
Through three kinds of substratum of mtt assay contrast, DMEM, DMEM-F12, α-MEM and serum-concentration 5 %, 10 %, three kinds of concentration culture effect of 15 % have drawn the multiplication culture that DMEM, 15 % serum-concentrations are fit to pig SSCs the most.According to bibliographical information, add non-essential amino acid; Beta-mercaptoethanol, L-glutaminate etc. are culturing cell bases, have played nutrition, the effect of anti-oxidant and energy matter.Shown in table one:
Different substratum of table 1 and serum-concentration are to the influence of pig SSCs proliferation rate
Notes: mark expression difference not remarkable (p＞0.05) together or with column data shoulder marking-up parent phase, alphabetical different table differential different significantly (p＜0.05).
According to table 1, two factor variance analytical resultss show, cultivate 3 d after, different substratum do not have the significance influence to pig SSCs proliferation rate, but after cultivating 5 d, DMEM and DMEM/F
12The pig SSCs proliferation rate of treatment group is significantly higher than α-MEM group, and after cultivating 7 d, the pig SSCs proliferation rate of DMEM treatment group is significantly higher than α-MEM group and DMEM/F
12Group; After cultivating 3 d, different serum-concentrations do not have the significance influence to pig SSCs proliferation rate yet, but after cultivating 5 d and 7 d, serum-concentration is that the pig SSCs proliferation rate of 15 % treatment group is significantly higher than 5 % and 10 % serum-concentration groups.The result of multiple comparisons shows, cultivate 3 d after, when serum-concentration is that 5 %, substratum are DMEM/F
12The time, pig SSCs proliferation rate is minimum; And after cultivating 5 d and 7 d, serum-concentration is 15 %, when substratum is DMEM, pig SSCs proliferation rate is the highest.It is thus clear that the DMEM culture system is the culture system that is more suitable for pig SSCs.
As a kind of preferred version, in the aforesaid method, the said nutrient solution staple that is used for culturing cell is: DMEM 83 %, L-glutaminate 1 %, non-essential amino acid 1 %, beta-mercaptoethanol 55 μ M, foetal calf serum 15 %.
After obtaining a large amount of stem spermatogonium cloning cluster through the inventive method; Through mechanical process picking cloning cluster, to new feeder layer, cultivation continues to go down to posterity through digestive inoculation; Or induce and be divided into other kind cells and be divided into sperm, also can be applied to induce experimental studies such as Ips cell; After obtaining a large amount of stem spermatogonium cloning cluster through the inventive method,, become unicellular back to carry out transgenic through electroporation through enzymic digestion through mechanical process picking cloning cluster.
Compared with prior art, the present invention has following beneficial effect:
The step enzyme digestion that we adopted is a kind of measure of incomplete digestion, is not to make whole testis tissue complete digestion become individual cells; But making convoluted tubule of testis be digested to the appearance that several cells are connected together, this possibly more meet stem spermatogonium and sustenticular cell interaction situation, possibly link to each other with several sustenticular cells by a stem spermatogonium; External cultivation under this condition; The sustenticular cell excretory factor has better promoted the propagation of early stage stem spermatogonium, obtains very pleasantly surprised of let us as a result, has seen that the whole plate form that all has been filled is good; The stem spermatogonium cloning cluster that refractivity is strong; And the two step enzyme methods of using always in the research, it is unicellular obtaining, and exist singly stem spermatogonium multiplication culture needs a large amount of factors to keep; But general cultivation effect can not reach the result that we obtain far away; So use a large amount of stem spermatogonium clone of the more effective acquisition of a step enzyme method, this method is efficiently a kind of, obtains the method for stem spermatogonium propagation fast.
Description of drawings
Fig. 1 shows sampling back inoculation 1 h, and cell attachment is fast, and state is good;
Fig. 2 observes for inoculation back the 2nd d, and cell enlargement is very fast, has formed a lot of decorative patterns, and there is the protuberance sign centre;
Fig. 3 is cell cultures 3 ~ 4 d, can see that extensive neat cell begins protuberance, and cloning cluster forms omen;
Fig. 4 produces for inoculation 5 ~ 7 d, a large amount of cloning cluster, neat in edge, and refractivity is good, covers with whole plate;
Fig. 5～6 are SEAP test positive synoptic diagram as a result;
Fig. 7 detects preceding original image for immunofluorescence;
Fig. 8 detects Hochest transfect cell nuclear figure for immunofluorescence;
Fig. 9 is an immunofluorescence Oct4 expression of results synoptic diagram;
Figure 10 is the RT-PCR Molecular Detection, and these cloning cluster are expressed the stem cell gene.
Embodiment
Come further to explain the present invention below in conjunction with embodiment, but embodiment does not do any type of qualification to the present invention.
The separation and the cultivation of embodiment 1 pig spermatogonium
(1) on super clean bench, the testis of piglet is put into the petridish that fills PBS, and, remove blood stains with PBS washing 2-3 times;
(2) remove albuginea testis, remove the reticular tissue in the parenchyma of testis, remaining testis tissue is cut into small pieces with iris, transfer in the 9 ml petridish with sharp tweezers;
(3) add the PBS of 10 times of volumes, blow and beat gently, leave standstill then with suction pipe, treat that testis tissue sinks to the bottom after, discard supernatant.So repetitive scrubbing is 2-3 time;
(4) collagenase digesting of 10 times of volumes of adding in 37 ℃ of water-bath 100 r/min, is blown and beaten with suction pipe, after can't see tissue block then gently.
(5) add 5-10 ml PBS, piping and druming gently, 16 ℃ then, centrifugal 5 min of 72 g (600 rpm) abandon supernatant, collect the bottom cell.
(6) cell counting.Using the outstanding cell adjustment of difference adherent culture basic weight cell concn is 5 * 10
6Individual/ml, be inoculated into 25 cm that prior usefulness 0.1 % gelatin encapsulates
2Culturing bottle is at 37 ° of C, 5 % CO
2, cultivate under the saturated humidity sterile culture case condition.
With pig SSCs to be detected, wash 1 time with PBS; Fixing 20 min under the 4 % Paraformaldehyde 96 room temperatures, PBS give a baby a bath on the third day after its birth time, each 5 min; Add 1 mL SEAP damping fluid, and add NBT 6.6 μ L and each 3.3 μ l of BCIP (vector), make a mixing head, fully mixing; Black out is hatched 10 ~ 15 min under the room temperature, cleans with PBS, and in time termination reaction is examined under a microscope and taken pictures (like Fig. 6).
Can judge tentatively that through AP dyeing the resulting cloning cluster of a step enzyme method is pig SSCs clone.
The identified by immunofluorescence of embodiment 3 pig SSCs detects
(1) after cell climbing sheet is made, takes out creep plate with 37 ℃ of PBS washings 3 times, each 3 ~ 5 s;
(2) with fixing 10 ~ 30 min of 4 % Paraformaldehyde 96s; PBS flushing 2 times, each 5 min;
(3) 0.5 % Triton perforation rupture of membranes is handled 15 min; PBS rinsing 2 times, each 5 min;
(4) 1 % BSA seal 30 min (sealed and need not wash);
(5) add one of 1 % BSA dilution and resist, spend the night in 4 ℃; PBS rinsing 2 times, each 5 min;
(6) add two of 1 % BSA dilution and resist, in 37 ℃ of reaction 1 h; PBS rinsing 2 times, each 5 min;
(7) 10 μ g/mL Hochest 5 min that dye; Anti-cancellation mounting is taken pictures.
Detect the resultant cloning cluster of a step enzyme method through immunofluorescence, its specifically expressing the important stem cell marker gene of Oct4, prove that it is a pig SSCs cloning cluster (Fig. 9).
To collect pig SSCs cloning cluster, clean 2 times centrifugal collecting cell with PBS.
1. total RNA extracts (according to TIANGEN company total RNA extraction reagent box specification sheets)
The detection of 2 total RNA samples
(1) total RNA integrity is through the sugared detected through gel electrophoresis of plain agar: prepare 1.5% sepharose: take by weighing 1.5 g agar Icing Sugar, be dissolved among 100 mL, 1 * TAE, boil in the microwave oven; Treat to add when solution is cooled to 50 ~ 60 ℃ EB; The perfusion gel, room temperature is placed 30 min, makes gelling solid; Application of sample electrophoresis: get the total RNA of about 4 ~ 5 μ L, add 3 μ L sample-loading buffers, mix the back and add in the point sample hole 5 V/cm voltage electrophoresis, 15 ~ 20 min; Electrophoresis finishes, and observes and takes pictures.
(2) concentration and purity detecting: get total RNA sample 1 μ L, dilute 50 times, measure the OD value in 260 nm and 280 nm wavelength with ultraviolet spectrophotometer.
3 total RNA reverse transcription methods are with reference to (the First strand cDNA synthetic agent box specification sheets that Japan is spun)
4 design of primers are with synthetic
According to the sequence that NCBI delivers about gene Oct4, Sox2, C-myc, Klf4, Nanog, Prdm14 and β-actin, adopt genetool software design primer, and give birth to worker Bioisystech Co., Ltd by Shanghai and synthesize, primer sequence information is seen table 3.
Table 2 primer sequence information
| Gene | Primer sequence (5-3 ') | Product (bp) | Annealing temperature (℃) | The Genebank sequence number |
| Oct4 | SEQ ID NO:1 | 521 | 59 | NM_001113060 |
| Sox2 | SEQ ID NO:2 | 604 | 55 | NM_001123197 |
| C-myc | SEQ ID NO:3 | 443 | 58 | NM_001005154 |
| Klf4 | SEQ ID NO:4 | 380 | 58 | NM_001031782 |
| Nanog | SEQ ID NO:5 | 614 | 61 | NM_001129971 |
| Prdm14 | SEQ ID NO:6 | 541 | 58 | XM_003125600 |
| β-actin | SEQ ID NO:7 | 612 | 59 | AY550069 |
5 pcr amplifications and product detect
Table 3 50 μ L reaction systems
The PCR response procedures is the preparatory sex change of 94 ℃/5 min; 94 ℃/30 s sex change, X ℃/30 s annealing, 72 ℃/45 s extend, and circulate 34 times; 72 ℃/10 min extend again, 10 ℃/10 min;
The PCR product detects: after PCR finishes; Get 6 μ L PCR products and 2 μ L bromjophenol blue mixing point samples; And with DNA Marker 2000 as reference; In 1 * TAE electrophoretic buffer, carry out 1.5 % sepharoses (containing 0.5 μ g/mL EB) electrophoresis (5 V/cm), electrophoresis is taken pictures with gel imaging system after finishing.
Detect a step enzyme method important stem cell marker gene such as Oct4, Sox2, C-myc, Klf4, Nanog, Prdm14 of having cultivated resulting cloning cluster specifically expressing through RT-PCR; And the cell that is used as feeder layer has confirmed further that from the molecule angle these cloning cluster are pig SSCs cloning cluster (like Figure 10) less than expressing.
SEQUENCE?LISTING
< 110>Agricultural University Of South China
< 120>a kind of method of a step enzyme method separation and Culture pig stem spermatogonium
<130>
<160> 7
<170> PatentIn?version?3.2
<210> 1
<211> 47
<212> DNA
<213> OCT-4
<400> 1
ggggctcact?ttgggggttc?tctcagggaa?tgggaccgag?gagtaca 47
<210> 2
<211> 41
<212> DNA
<213> SOX-2
<400> 2
cggcggtggc?aactctactg?gggcgagccg?ttcatgtagg?t 41
<210> 3
<211> 38
<212> DNA
<213> C-MYC
<400> 3
gcgggcacgg?cggctactgg?ggggcgctgc?ataattgt 38
<210> 4
<211> 39
<212> DNA
<213> KLF-4
<400> 4
cgcgcatgtg?ccccaagatc?ccggggccac?gaccttctc 39
<210> 5
<211> 47
<212> DNA
<213> Nanog
<400> 5
cttattcagg?acagccctga?ttcttcaaga?cggcctccaa?atcactg 47
<210> 6
<211> 46
<212> DNA
<213> PRDM-14
<400> 6
cgcccccagt?ggatgcttct?ctcgggcaca?gttgacatag?gacatc 46
<210> 7
<211> 50
<212> DNA
<213> βaction
<400> 7
ccgtgagaag?atgacccaga?tcatgtcgtg?atctccttct?gcatcctgtc 50
Claims (3)
1. the method for a step enzyme method separation and Culture pig stem spermatogonium is characterized in that comprising the steps:
(1) testis of piglet is put into the container that fills PBS,, remove blood stains with PBS washing 2 ~ 3 times;
(2) remove albuginea testis and reticular tissue, remaining testis tissue is cut into small pieces, transfer in the petridish;
(3) add the PBS of 10 times of volumes,, leave standstill with suction pipe piping and druming, treat that testis tissue sinks to the bottom after, abandon supernatant;
(4) repeating step is (3) 2 ~ 3 times;
(5) collagenase digesting of 10 times of volumes of adding is in 37 ℃ water-baths, 100 rpm, then with suction pipe piping and druming, up to can't see tissue block;
(6) PBS of adding 5 ~ 10 ml, piping and druming at 16 ℃, centrifugal 5 min of 600 rpm, is abandoned supernatant, collects the bottom cell;
(7) with the outstanding cell of difference adherent culture basic weight, the adjustment cell concn is 5 * 10
6Individual/ml, be inoculated in the culturing bottle that prior usefulness 0.1 % gelatin encapsulates, in the sterile culture case, cultivate.
2. the method for a step enzyme method separation and Culture pig stem spermatogonium according to claim 1, the culture condition that it is characterized in that said cell culture incubator is 37 ℃, 5 % CO
2, saturated humidity.
3. the method for a step enzyme method separation and Culture pig stem spermatogonium according to claim 1; It is characterized in that the said nutrient solution staple that is used for culturing cell is: DMEM 83 %, L-glutaminate 1 %, non-essential amino acid 1 %; Beta-mercaptoethanol 55 μ M, foetal calf serum 15 %.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103805560A (en) * | 2014-01-20 | 2014-05-21 | 广西大学 | Method for separating pig spermatogonia stem cells |
| CN108795850A (en) * | 2018-06-26 | 2018-11-13 | 西北农林科技大学 | A kind of Spermatogonial Stem Cells are without feeder layer long-period culture method |
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| KR20090013543A (en) * | 2007-08-02 | 2009-02-05 | 건국대학교 산학협력단 | Method for isolating spermatogonial stem cell |
| CN101818127A (en) * | 2010-03-31 | 2010-09-01 | 安徽农业大学 | Method for separating and culturing mouse primitive spermatogonia |
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2011
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| KR20090013543A (en) * | 2007-08-02 | 2009-02-05 | 건국대학교 산학협력단 | Method for isolating spermatogonial stem cell |
| CN101818127A (en) * | 2010-03-31 | 2010-09-01 | 安徽农业大学 | Method for separating and culturing mouse primitive spermatogonia |
Non-Patent Citations (2)
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| 宋锐 等: "一种简便的小鼠精原干细胞分离培养方法", 《中国细胞生物学学报》 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103805560A (en) * | 2014-01-20 | 2014-05-21 | 广西大学 | Method for separating pig spermatogonia stem cells |
| CN103805560B (en) * | 2014-01-20 | 2015-11-04 | 广西大学 | The separation method of one boar stem spermatogonium |
| CN108795850A (en) * | 2018-06-26 | 2018-11-13 | 西北农林科技大学 | A kind of Spermatogonial Stem Cells are without feeder layer long-period culture method |
| CN108795850B (en) * | 2018-06-26 | 2021-08-31 | 西北农林科技大学 | Long-term culture method for spermatogonial stem cells without feed layer in vitro |
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