CN102824226B - A kind of preparation method of spermatogonial stem cell transplantation receptor - Google Patents
A kind of preparation method of spermatogonial stem cell transplantation receptor Download PDFInfo
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- CN102824226B CN102824226B CN201210343103.9A CN201210343103A CN102824226B CN 102824226 B CN102824226 B CN 102824226B CN 201210343103 A CN201210343103 A CN 201210343103A CN 102824226 B CN102824226 B CN 102824226B
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Abstract
The invention discloses a kind of method prepared by spermatogonial stem cell transplantation receptor, the method prepares spermatogonial stem cell transplantation receptor by testis injection busulfan.Application the inventive method, not only successfully can prepare spermatogonial stem cell transplantation receptor fast, reduces the comparatively major injury that lumbar injection busulfan method causes animal bone marrow, hemopoietic system, reduces or avoid animal dead, can also reduce drug use amount, reduce costs.This method has hewed out a kind of novel spermatogonial stem cell transplantation receptor preparation method.The method does not relate to any instrumentation, existing conventional instrument and chemical reagent is only utilized to complete, one of ordinary skill in the art all can implement the present invention, a kind of with low cost, easy and simple to handle, efficient, quick, safe, feasible spermatogonial stem cell transplantation receptor preparation method, contributes to applying and innovate and optimize of spermatogonial stem cell transplantation technology.
Description
Technical field
The invention belongs to applied biological technical field; a kind of spermatogonial stem cell transplantation receptor preparation method of novel, efficient, low toxicity, can at spermatogonial stem cell transplantation, utilize stem spermatogonium to prepare transgenic animal and utilize stem spermatogonium to protect in the fields such as Endangered species and apply.
Background technology
Stem spermatogonium is the diploid cell that a group of the nearly basement membrane of spermatogenic epithelium has height self renewal and multi-lineage potential, is uniquely hereditary information can be passed to follow-on adult stem cell in adults body.Stem spermatogonium can continue self renewal and keep self Population constant, constantly can break up again and produce spermatogonium at different levels, and finally generate sperm.The research of foundation to spermatogenesis mechanism of spermatogonial stem cell transplantation system serves impetus, also for treatment male sterility, exploitation male contraception medicine, the fertility of preserving tumor or perch roadway patient, protection animals on the brink of extinction, promote good quality and high output animal breeding and transgenic animal to prepare etc. provide possibility.Now, successfully achieve receptor preparation and the transplanting of the species stem spermatogoniums such as mice, rat, bull, goat, pig, the research especially on mice is more ripe.
Receptor preparation is the important component part of spermatogonial stem cell transplantation technology, and be essential condition and the antecedent basis of successful implantation, efficiency prepared by receptor is directly connected to transplantation effect.At present, lumbar injection busulfan, local roentgenization, local heat treatmet etc. are established by Antibody Production Techniques, by these technical finesses, make the spermatogenic cells at different stages generation apoptosis of receptor testis, cavity is formed in the nearly chamber cell and basilar compartment of the encirclement of convoluted seminiferous tubule sustenticular cell, be convenient to transplant moving to the interior and attachedly planting of rear donor stem spermatogonium, and then complete regeneration differentiation and spermatogenesis.Although established comparatively ripe by preparation, preparation process is comparatively loaded down with trivial details, and preparation time is longer, and drug injection easily causes receptor dead, and whole efficiency is lower.
Lumbar injection busulfan method
Busulfan is a kind of antineoplastic agent of antiproliferative effect, can make DNA alkylation thus the increment of destruction receptor stem spermatogonium.Busulfan can remove most intermediate cell in most stem spermatogonium in animal testis and spermatogenesis, be use the earliest by preparation.But busulfan can produce inhibitory action to the bone marrow of animal, hemopoietic system, damages very large.When receptor prepared by lumbar injection busulfan, the mortality rate that laboratory animal is higher can be caused.Also have experiment to adopt injection of scrotum to prepare receptor, although reduce the injury to laboratory animal, experiment effect is still not bery desirable.
Local roentgenization method
Local roentgenization, is use various dose X-ray to irradiate animal part, under the prerequisite damaging other organ cells hardly, makes spermatogonium at different levels in receptor testis be subject to destroying comparatively thoroughly.This method is more suitable for large animal, although directly can kill spermatogonium, needs special irradiation apparatus, uses inconvenience, and equipment costly, complicated operation, dosage not easily determines.Also can cause radioactivity pollution in experiment, and there is the potential safety hazard making receptor lethal equally.
Heat treating process
Compared with first two method, process effect can be confined to target spot position by heat treating process better, weakens the seondary effect to other positions of health.Research from Mouse and rat shows, 42-43 DEG C of high temperature can cause apoptosis of germ cells, and heat treatment can cause testicular weight to reduce in a short time, and then causes the of short duration slightly sterile or completely sterile of process Mus.But the receptor testis microenvironment of this method process recovers very fast, and transplant and will could obtain the highest transplanting efficiency in the best holds the phase, the necessary rigorous concentration transplant time of the receptor therefore using this legal system standby, otherwise transplanting result can be affected.Although heat treating process is to animal nonhazardous effect, its effect is relatively not obvious, requires strict, less use to the assurance of time.
In recent years, animal welfare and receptor safety are more and more subject to the attention of industry, make to have more challenge by preparation, therefore, are necessary to set up a kind of simple, safety, efficient novel spermatogonial stem cell transplantation receptor technology of preparing.
Summary of the invention
The object of the invention is to provide a kind of method preparing spermatogonial stem cell transplantation receptor for above-mentioned deficiency.
For achieving the above object, the present invention adopts following technical scheme:
Prepare a method for spermatogonial stem cell transplantation receptor, the method busulfan is injected receptor testis to prepare transplant recipient.
Preferably, make syringe needle thrust testis to long axis direction far-end along testis long axis direction during injection, then while medicine is pushed, syringe needle is slowly extracted out, busulfan is injected in the convoluted seminiferous tubule of each lobule of testis.
The injected dose of busulfan can be determined according to different plant species, and with 3.5 ~ 4.5mg/ml concentration, it is good that the dosage of 3 ~ 5mg/kg body weight injects Recipient mice testis, within 21 ~ 28 days, obtains transplant recipient after process.Better injection system is by busulfan with a 4mg/ml concentration, and the dosage of 4mg/kg body weight injects mouse receptor testis, within 21 ~ 28 days, obtains transplant recipient after process.
Based on this, the present invention also provides a kind of reagent for the preparation of spermatogonial stem cell transplantation receptor, and it is the busulfan solution of 3 ~ 5mg/ml.
Instrument for injecting comprises injector syringe and injection needle, with hose connection between injector syringe and injection needle.The syringe that such as can making connects connects and venoclysis needle.Such as adopt 0.45mm × 15mm specification disposable venous infusion needle to be connected with syringe particularly and make injection tool.According to the difference of receptor, accommodation can be carried out to the specification of syringe needle.
Utilize the present invention, the injury to animal bone marrow, hemopoietic system can be greatly reduced, reduce or avoid the death of receptor.Because directly carry out drug injection to target organ, enable medicine play drug effect with the fastest speed, and decrease the injury to other organs, thus greatly reduce and even avoid receptor death.
After mouse testis injection busulfan, occur a large amount of cavity in convoluted seminiferous tubule, the compact siro spinning technology simultaneously between sustenticular cell is destroyed, for transplanting moving to the interior and attachedly planting the condition of providing of rear donor stem spermatogonium.Process 21-28d, the blank pipe in convoluted seminiferous tubule reaches at utmost, and the compact siro spinning technology subsequently between sustenticular cell progressively recovers.Therefore, after selecting process, the mouse receptor of 21d-28d is transplanted.
The present invention, by changing traditional busulfan injection site, reduces and even avoids receptor death; By improving injection needle, to be connected with syringe by the disposable venous infusion needle of 0.45mm × 15mm specification and to substitute syringe needle and draw pin syringe needle, both reduced to inject technical difficulty is again reduced to the wound of testis.The present invention does not relate to any instrumentation, and only utilize existing conventional instrument and chemical reagent to complete, reduce preparation cost, in addition, one of ordinary skill in the art all can implement the present invention.
The present invention can realize the efficient preparation of spermatogonial stem cell transplantation receptor, for the rapid raising of spermatogonial stem cell transplantation technology and the exploration of new method provide simple, safe, feasible technical support, and contribute to application prepared by stem spermatogonium receptor and popularization, thus promote the application of spermatogonial stem cell transplantation technology.
Accompanying drawing explanation
Fig. 1 is the section of different disposal group mouse testis
Wherein, A, untreated fish group normal testis section H.E colored graph.B(B1, B2, B3, B4), 4mg/kg/ side dosage busulfan testis injection group receptor is at 14d, 21d, 28d, 35d testis section H.E colored graph.C(C1, C2, C3), for 40mg/kg dosage busulfan lumbar injection group receptor is at 14d, 21d, 28d testis section H.E colored graph.(amplification: 400X)
Fig. 2 is the section of different injected dose group mouse testis
Wherein, A(A1, A2, A3, A4) 2mg/kg/ side dosage group, 14d, 21d, 28d, 35d mouse testis section H.E colored graph after process.B(B1, B2, B3, B4) 3mg/kg/ side dosage group, 14d, 21d, 28d, 35d mouse testis section H.E colored graph after process.C(C1, C2, C3, C4) 4mg/kg/ side dosage group, 14d, 21d, 28d, 35d week mouse testis section H.E colored graph after process.(amplification: 400X).
Specific implementation method
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.
Prepared by embodiment 1 receptor
Receptor: 1-2 monthly age CD1 (ICR) strain normally breeds public Mus.Purchased from Beijing Vital River Experimental Animals Technology Co., Ltd..
Configuration busulfan: busulfan is dissolved in DMSO, and concentration is 8mg/ml, injects first 0.5 hour, adds equal-volume ultra-pure water, and determining final concentration is 4mg/ml, 37 DEG C of water-baths, Anti-solidification.
Injection: be connected for subsequent use with syringe by the disposable venous infusion needle of 0.45mm × 15mm specification, injection concentration 4mg/ml, dosage is respectively 2mg/kg, 3mg/kg, 4mg/kg, 5mg/kg, and injection volume is 1/10 of lumbar injection.Set up lumbar injection matched group and blank group, lumbar injection group carries out lumbar injection in conventional manner simultaneously, and injected dose is 40mg/kg.
Section: busulfan testis injection group receptor after injection 14d, 21d, 28d, 35d time get respectively various dose group and contrast make testis section, H.E dye.
Result: as depicted in figs. 1 and 2, can in the minimizing causing spermatogenic cells at different stages in varying degrees by testis injection, wherein with the effect of 3 ~ 5mg/kg/ side comparatively obvious (5mg/kg/ side is not shown), and 4mg/kg/ side is the optimal dose that receptor prepared by testis injection busulfan.After mouse testis injection busulfan, along with the prolongation of time after process, the spermatogenic cells at different stages in convoluted seminiferous tubule reduces gradually, reduce to minimum at 21-28d, convoluted seminiferous tubule hollow, spermatogenic cells at different stages disappears, recover gradually afterwards, 35d can see in convoluted seminiferous tubule and occur a large amount of sexual cell.The mice of lumbar injection busulfan is along with the prolongation processing the rear time, and also show spermatogenic cells at different stages in testis and start to reduce, 21d reduces to minimum, the process recovered gradually afterwards.But can be found out by section, the damage of testis injection busulfan to testis structure is less, and the time that convoluted seminiferous tubule hollow continues is longer.
Table 1 is the dead mouse statistical result of different disposal group
| Grouping | Treatment dosage | Treating number/only | Mortality/only | Mortality rate/% |
| Lumbar injection group | 40mg/kg | 95 | 34 | 35.8 |
| Testis injection group | 4mg/kg/ side | 55 | 0 | 0 |
| Blank group | 0/kg/ side | 50 | 0 | 0 |
In addition, compared with lumbar injection group, testis injection avoids the phenomena of mortality because drug injection causes, and improves receptor preparation efficiency, reduces cost.
Embodiment 2 spermatogonial stem cell transplantation
Donor: the public Mus of 4-6 age in days C57 red fluorescence.Purchased from Institute of Experimental Animals, Chinese Academy of Medical Sciences's heredity center
Prepared by donorcells suspension
Two step enzyme digestions are adopted to prepare cell suspension.Take out the C57 red fluorescence mice of raw rear 4-6 age in days, put to death, take out testis and put into fast without calcium magnesium PBS.10-20 testis is got in each test, removes tunica albuginea, splits, spreads convoluted seminiferous tubule out, and visible cell silk ribbon between removing tubule, puts into centrifuge tube shears and shred.Add Collagenase 1ml(1mg/ml), DNase(0.4mg/ml) digest 5min, and continue piping and druming, the centrifugal 3min of 1000r/min.Supernatant discarded, adds 1ml PBS and cleans, and centrifugal 3-4min after mixing, repeats 2 times.Supernatant discarded, adds trypsinization 3min, adds culture medium 500-1000 μ l and stops digestion, centrifugal 4-5min.With PBS cleaning 2-3 time, supernatant discarded, adds culture fluid.
Be filled into 400 order cell sieves in the culture dish being covered with 0.2% (w/v) gelatin, the adherent sorting cells of difference.At 37 DEG C, 5%CO
2under condition, cultivate 40 minutes to 2 hours, the time depends on cell attachment situation, most of adherent and stem spermatogonium is loosely to be attached on sustenticular cell layer as well with sustenticular cell.Blow and beat gently with suction pipe, hanged not yet closely adherent stem spermatogonium, according to 10
6-10
7/ ml concentration Eddy diffusion, in centrifuge tube, is placed in for subsequent use on ice.
Spermatogonial stem cell transplantation
After utilizing embodiment 1 testis busulfan (4mg/kg) to process, the Recipient mice of 21d-28d carries out transplantation experiments.Donor stem spermatogonium after sorting is implanted in Recipient mice testis through ductuli efferentes testis, co-transplantation 10 receptors.After transplanting 45d, receptor Mus and the female Mus of ICR are mated, obtain 2 nest offsprings.
Claims (5)
1. prepare the method for spermatogonial stem cell transplantation receptor for one kind, the method busulfan is injected receptor testis to prepare transplant recipient, injection needle is made to thrust testis to long axis direction far-end along testis long axis direction during injection, then while medicine is pushed, syringe needle is slowly extracted out, busulfan is injected in the convoluted seminiferous tubule of each lobule of testis.
2. method according to claim 1, is characterized in that, the method is by busulfan with 3.5 ~ 4.5mg/ml concentration, and the dosage of 3 ~ 5mg/kg body weight injects receptor testis, within 21 ~ 28 days, obtains transplant recipient after process.
3. method according to claim 2, is characterized in that, the method is by busulfan with 4mg/ml concentration, and the dosage of 4mg/kg body weight injects receptor testis, within 21 ~ 28 days, obtains transplant recipient after process.
4. method according to claim 1 and 2, is characterized in that, implantation tool comprises injector syringe and injection needle, with hose connection between injector syringe and injection needle.
5. method according to claim 4, is characterized in that, described implantation tool is the connect injector syringe and venoclysis needle that connect.
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| CN102105579A (en) * | 2007-08-07 | 2011-06-22 | 普里梅真比奥特斯公司 | Isolation, characterization and propagation of germline stem cells |
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| 《Culture of mouse spermatogonial stem cells》;Makoto Nagano 等;《Tissue & Cell》;19981231;第30卷(第4期);389-397 * |
| 《化疗药物建立小鼠无精子症模型的实验研究》;李智会等;《吉林医药学院学报》;20090630;第30卷(第3期);138-143 * |
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