CN102417893B

CN102417893B - Method for separating and culturing swine spermatogonial stem cells by using one-step enzyme process

Info

Publication number: CN102417893B
Application number: CN 201110272927
Authority: CN
Inventors: 张守全; 白银山
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2011-09-15
Filing date: 2011-09-15
Publication date: 2013-07-31
Anticipated expiration: 2031-09-15
Also published as: CN102417893A

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

A kind of method of a step enzyme method separation and Culture pig stem spermatogonium

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

Stem spermatogonium (Spermatogonialstemcells, SSCs) be uniquely in the male adult of mammal genetic information can be passed to follow-on stem cell, 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 transgenosis, and it generates sperm, finish the transmission of gene, be accompanied by the rise of transgenic technology, it becomes a kind of new transgenosis approach.

Spermatogeny is the process of a complexity, 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 the source that stem spermatogonium is transplanted for cell replacement therapy and histoorgan from now on becomes possibility.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 by the following technical programs:

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 culture dish;

(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-bath 100rpm, then with suction pipe piping and druming, up to can't see tissue block;

(6) PBS of adding 5～10ml, piping and druming at 16 ℃, the centrifugal 5min of 600rpm, is abandoned supernatant, collects the bottom cell;

(7) with the outstanding cell of difference adherent culture basic weight, adjusting cell concn is 5 * 10 ⁶Individual/ml, be inoculated in the culturing bottle of prior usefulness 0.1% gelatin bag quilt, in the sterile culture case, cultivate.

As a kind of preferred version, in the aforesaid method, the culture condition of described cell culture incubator is 37 ℃, 5%CO2, saturated humidity.

By three kinds of substratum of mtt assay contrast, DMEM, DMEM-F12, α-MEM and 5%, 10%, 15% 3 kind of concentration culture effect of serum-concentration have drawn the multiplication culture that DMEM, 15% serum-concentration are fit to pig SSCs the most.According to bibliographical information, add non-essential amino acid; Beta-mercaptoethanol, L-glutaminate etc. are the culturing cell bases, have played nutrition, the effect of anti-oxidant and energy matter.As shown in Table 1:

Different substratum of table 1 and serum-concentration are to the influence of PSSCs 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 that behind the cultivation 3d, different substratum do not have the significance influence to the PSSCs proliferation rate, but after cultivating 5d, DMEM and DMEM/F ₁₂The PSSCs proliferation rate of treatment group is significantly higher than α-MEM group, and after cultivating 7d, the PSSCs proliferation rate of DMEM treatment group is significantly higher than α-MEM group and DMEM/F ₁₂Group; After cultivating 3d, different serum-concentrations do not have the significance influence to the PSSCs proliferation rate yet, but after cultivating 5d and 7d, serum-concentration is that the PSSCs proliferation rate of 15% treatment group is significantly higher than 5% and 10% serum-concentration group.The result of multiple comparisons shows, cultivate 3d after, when serum-concentration is 5%, substratum is DMEM/F ₁₂The time, the PSSCs proliferation rate is minimum; And after cultivating 5d and 7d, serum-concentration is 15%, when substratum is DMEM, the PSSCs proliferation rate is the highest.As seen the DMEM culture system is the culture system that is more suitable for PSSCs.

As a kind of preferred version, in the aforesaid method, the described nutrient solution main component that is used for culturing cell is: DMEM83%, 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 by the inventive method, by mechanical process picking cloning cluster, to new feeder layer, cultivation continues to go down to posterity by 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 by the inventive method,, become unicellular back to carry out transgenosis by electroporation through enzymic digestion by mechanical process picking cloning cluster.

Compared with prior art, the present invention has following beneficial effect:

The step enzyme digestion that we adopted, it is a kind of measure of incomplete digestion, not to make whole testis tissue complete digestion become individual cells, but make convoluted tubule of testis be digested to the appearance that several cells are connected together, this may more meet stem spermatogonium and sustenticular cell interaction situation, may 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, obtain very pleasantly surprised of let us as a result, 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 Individual existence 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 1h, and cell attachment is fast, and state is good;

Fig. 2 observes for inoculation back 2d, 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 3～4d, can see that extensive neat cell begins protuberance, and cloning cluster forms omen;

Fig. 4 produces for inoculation 5～7d, a large amount of cloning cluster, neat in edge, and refractivity is good, covers with whole plate;

Fig. 5～6 are alkaline phosphatase test positive result schematic diagram;

Fig. 7 detects preceding original image for immunofluorescence;

Fig. 8 detects Hochest transfect cell nuclear figure for immunofluorescence;

Fig. 9 is an immunofluorescence Oct-4 expression of results synoptic diagram;

Figure 10 is the RT-PCR Molecular Detection, and these cloning cluster are expressed the stem cell gene.

Embodiment

Further 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, piglet testis is taken out in operation, puts into the culture dish that fills PBS, and with PBS washing 2-3 time, removes blood stains;

(2) remove albuginea testis, remove reticular tissue in the parenchyma of testis, remaining testis tissue is cut into small pieces with iris, transfer in the 9ml culture dish 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 liquor.So repetitive scrubbing is 2-3 time;

(4) collagenase digesting of 10 times of volumes of adding in 37 ℃ of water-bath 100r/min, is blown and beaten with suction pipe, after can't see tissue block then gently.

(5) add 5-10mlPBS, piping and druming gently, 16 ℃ then, the centrifugal 5min of 72g (600rpm) abandons supernatant, collects the bottom cell.

(6) cell counting.Adjusting cell concn with the outstanding cell of difference adherent culture basic weight is 5 * 10 ⁶Individual/ml, be inoculated into the 25cm of prior usefulness 0.1% gelatin bag quilt ²Culturing bottle is at 37 ° of C, 5%CO ₂, cultivate under the saturated humidity sterile culture case condition.

Embodiment 2 alkaline phosphatases (AlkalinePhosphatase, AP) positive detection

With PSSCs to be detected, wash 1 time with PBS; Fixing 20min under the 4% Paraformaldehyde 96 room temperature, PBS give a baby a bath on the third day after its birth time, each 5min; Add 1mL alkaline phosphatase damping fluid, and add NBT6.6 μ L and BCIP(vector) each 3.3 μ l, make a mixing tube, fully mixing; Black out is hatched 10～15min under the room temperature, cleans with PBS, and in time termination reaction is examined under a microscope and taken pictures (as Fig. 6).

Can judge tentatively that by AP dyeing the resulting cloning cluster of a step enzyme method is the PSSCs clone.

The identified by immunofluorescence of embodiment 3 pig PSSCsSSCs detects

(1) after cell climbing sheet is made, takes out creep plate and wash 3 times each 3～5s with 37 ℃ of PBS;

(2) fix 10～30min with 4% Paraformaldehyde 96; PBS flushing 2 times, each 5min;

(3) 0.5%Triton perforation rupture of membranes is handled 15min; PBS rinsing 2 times, each 5min;

(4) 1%BSA sealing 30min(has sealed and need not wash);

(5) add one of 1%BSA dilution and resist, spend the night in 4 ℃; PBS rinsing 2 times, each 5min;

(6) add two of 1%BSA dilution and resist, in 37 ℃ of reaction 1h; PBS rinsing 2 times, each 5min;

(7) the 10 μ g/mLHochest 5min that dyes; Anti-cancellation mounting is taken pictures.

Detect the resultant cloning cluster of a step enzyme method by immunofluorescence, its specifically expressing the important stem cell marker gene of Oct4, prove that it is PSSCs cloning cluster (Fig. 9).

Embodiment 4 carries out Molecular Detection by RT--PCR and identifies SSCs

To collect the PSSCs 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 by the sugared detected through gel electrophoresis of plain agar: prepare 1.5% sepharose: take by weighing 1.5g agar Icing Sugar, be dissolved among 100mL1 * TAE, boil in the microwave oven, treat to add when solution is cooled to 50～60 ℃ EB, the perfusion gel, room temperature is placed 30min, 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 5V/cm voltage electrophoresis 15～20min; 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 260nm and 280nm wavelength place with ultraviolet spectrophotometer.

3 total RNA reverse transcription methods are with reference to (the FirststrandcDNA 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 sees Table 3.

Table 2 primer sequence information

The 5PCR amplification detects with product

Table 350 μ L reaction system

The PCR response procedures is the pre-sex change of 94 ℃/5min; 94 ℃/30s sex change, X ℃/30s annealing, 72 ℃/45s extend, and circulate 34 times; 72 ℃/10min extends again, 10 ℃ of 10min;

The PCR product detects: after PCR finishes, get 6 μ LPCR products and 2 μ L bromjophenol blue mixing point samples, and with DNAMarker2000 as reference, in 1 * TAE electrophoretic buffer, carry out 1.5% sepharose (containing 0.5 μ g/mLEB) electrophoresis (5V/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 by RT-PCR, and the cell that is used as feeder layer has confirmed further that from the molecule angle these youngster's cloning cluster are PSSCs cloning cluster (as 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

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;

(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, adjusting cell concn is 5 * 10 ⁶Individual/ml, be inoculated in the culturing bottle of prior usefulness 0.1 % gelatin bag quilt, 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 described 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 described nutrient solution main component 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 %.