CN102690782A - Fibroblast culture solution - Google Patents

Abstract

The invention discloses a fibroblast culture solution. The fibroblast culture solution comprises the following components: 10% of fetal bovine serum, 4ng/ml of basic fibroblast growth factor, 2.5mu g/ml of Plasmocin, 100U/ml of penicillin and 100mu g/ml of streptomycin, and a high glucose DMEM (Dulbecco minimum essential medium) culture solution is made up to 100%. Compared with the conventional fibroblast culture solution, the fibroblast culture solution adopting the design disclosed by the invention can realize fast fibroblast growth speed and strong proliferation ability, and a cultured fibroblast has the typical fibrous form and stronger anti-apoptotic ability and is less prone to aging. Therefore, the fibroblast cultured by the fibroblast culture solution adopting the design disclosed by the invention can be used for producing cell engineering products with higher quality and higher efficiency by being applied to production practices.

Description

A kind of inoblast nutrient solution

 

Technical field

The present invention relates to the animal cell culture technical field, relate in particular to a kind of inoblast nutrient solution.

Background technology

Cell is substruction and the functional unit of organism; In the current society that life science develops rapidly, great scientific advancees such as animal cloning, stem cell, galactophore biological reactor transgenic animal, artificial tissue and organ all be unable to do without the platform that cell engineering is set up.Cell engineering is human according to the certain designed scheme, through on cell, ubcellular or tissue level, carrying out experimental implementation, obtains cell, tissue, the organ of reconstruct, even individual, thereby cultivates the comprehensive biotechnology of creating outstanding kind or product.Aspects such as the related major technique field of current cell engineering has cell cultures, cytogamy, cell to tear open to close, karyomit(e) operation and transgenosis.Cell engineering has been penetrated into many fields such as human lives's clothing, food, lodging and transportion--basic necessities of life, like the medicine of treatment disease, prophylactic vaccine, the makeup of beauty treatment, the food of eating etc.Cell cultures is platform and the basis that cell engineering is able to commercial application, and a large amount of cells is used for producing and research just can be produced the cell engineering product through turning out.In cultivating numerous cells of using, inoblast is one of most widely used cell.Inoblast has wide material sources, gains the name because of cell presents fibrous form, all contains a large amount of inoblasts in the various histoorgans, and inoblast is usually used in the quick reparation of damaged tissue organ in tissue.At present, inoblast has been widely used in great scientific research and production practice such as animal cloning, stem cell, transgenic galactophore biological reactor animal, artificial tissue and organ.But the inoblast of high quality and growth performance is most important for the quality and the efficient of cell engineering product.

In fibroblastic incubation growth process, cell culture fluid is absolutely necessary.Present conventional cell culture fluid in use exists fibroblastic growth speed slow, and a little less than the multiplication capacity, the inoblast of cultivation does not often have typical fibrous form, and anti-apoptosis capacity is more weak and the problem of easy aging.

Summary of the invention

The technical problem that the present invention will solve provides a kind of inoblast nutrient solution.

In order to solve the problems of the technologies described above, the technical scheme that the present invention adopts is: a kind of inoblast nutrient solution, composed of the following components:

10% foetal calf serum, 4 ng/ml Prostatropins, 2.5 μ g/ml Plasmocin, 100 U/ml penicillium mould, 100 μ g/ml Streptomycin sulphates, high sugared DMEM nutrient solution supplies 100%.

(basic fibroblast growth factor bFGF) is fibroblast growth factor albumen (fibroblast growth factors, FGFs) a member in the family to Prostatropin.Found at least 22 kinds of FGF at present, bFGF is a kind of very active substance of trace in Mammals and the human body, has physiological function and important clinical application value widely.BFGF has can stimulate new vessel to form, and participates in wound healing and tissue regeneration, promotes embryonic tissue growth and differentiation etc., closely related with the incidence and development of tumour in addition.In recent years, recombinant bfgf has been applied to diseases such as clinical treatment wound, ulcer and neural system.

The mycoplasma infection incidence reaches 63% in cell cultures, and mycoplasma infection can change DNA, RNA and the protein expression of cell after taking place, thereby causes cell poor growth and second-rate.Plasmocin belongs to sterilization microbiotic of new generation, can act on the cell that infects mycoplasma by intensive, kills mycoplasma.Simultaneously; Plasmocin can bring into play the resisting gram-positive and the gram-negative bacteriological action of wide spectrum with a kind of lower activity; Such as the bacterium that can act on penicillin resistant and Streptomycin sulphate, confirmed that Plasmocin does not have toxicity to eukaryotic cell, do not influence the metabolism of cell itself.

The invention has the beneficial effects as follows:

The inoblast nutrient solution of the present invention's design is compared with conventional inoblast nutrient solution; Can make fibroblastic growth speed fast; Multiplication capacity is strong, and the inoblast of cultivation has typical fibrous form, and has stronger anti-apoptosis capacity and difficult aging.Therefore, the inoblast cultivated of the inoblast nutrient solution of the present invention design is applied to production practice and will produces the more cell engineering product of high quality and efficient.

Description of drawings

Below in conjunction with accompanying drawing and embodiment the present invention is done further detailed explanation.

Fig. 1 is fibrous cell's form of in the inoblast nutrient solution of the present invention's design, growing.

Fig. 2 is fibrous cell's form of in conventional inoblast nutrient solution, growing.

Embodiment

One, materials and methods

1, fibroblastic cultivation

Getting the inoblast of ox, sheep, pig and mouse cultivates; Experimental group adopts the novel fibroblast cell culture fluid (calling novel nutrient solution in the following text) of design, and the component of novel nutrient solution is: 10% FBS+4 ng/ml Prostatropin+2.5 μ g/ml Plasmocin+100 U/ml penicillium mould+100 μ g/ml Streptomycin sulphates+high sugared DMEM nutrient solutions.Control group adopts conventional inoblast nutrient solution (calling conventional nutrient solution in the following text), and the component of conventional nutrient solution is: 10% FBS+100 U/ml penicillium mould+100 μ g/ml Streptomycin sulphates+high sugared DMEM nutrient solution.Get the inoblast of the identical cultivation algebraically of different plant species respectively, adopt above-mentioned two kinds of nutrient solutions to cultivate.

2, the fibroblasts proliferation ability detects

Inoculate the inoblast of different plant species respectively by identical density; Adopt above-mentioned two kinds of nutrient solutions; Under the identical culture condition of other factors, cultivate,, fix with 4% Paraformaldehyde 96 for the cell that the growing multiplication ability detects; Carry out nucleus dyeing with the optical dye DAPI of 1 μ g/ml, observation of cell propagation result and proliferative activity under the fluoroscope behind the 1min.Simultaneously, use trypsin digestion cell, collect individual cells, carry out cell counting, the multiplication capacity of statistical study cell.

3, fibroblastic anti-apoptosis capacity detects

By the inoblast of identical density inoculation different plant species, adopt above-mentioned two kinds of nutrient solutions respectively, under the identical culture condition of other factors, cultivate; MTC with 10 μ g/ml is processed into fibrocyte; Be digested to fibrocyte after the processing, inoculate and cultivate, respectively at fixing with 4% Paraformaldehyde 96 after 1 week; Optical dye DAPI with 1 μ g/ml carries out nucleus dyeing, the anti-apoptosis capacity of observation of cell under the fluoroscope behind the 1min.Under situation about handling without MTC; Pair cell goes down to posterity respectively; Fix with 4% Paraformaldehyde 96 second day of cell growth when being passaged to for the 5th generation, carry out the nucleus anti-apoptosis capacity of observation of cell under the fluoroscope that dyes behind the 1min with the optical dye DAPI of 1 μ g/ml.

Two, experimental result

1, fibroblastic typical fibers shape growthhabit

As shown in Figure 1; After the novel nutrient solution that uses design; The inoblast of ox, sheep, pig and mouse all presents typical fibrous growthhabit, and its fibrous form of inoblast that is in growth phase is comparatively short and thick, and the inoblast that is in the confluent culture ware stage then presents long and narrow fibrous form; Connect between the inoblast closely, unit cell growth area cell density is bigger.Fibrous cell closely is connected to form vortex shape cell colony form, and vortex shape cell colony form is the fibroblastic important cells form performance of high quality.As shown in Figure 2, under identical culture environment condition, use the inoblast of ox, sheep, pig and the mouse of conventional nutrient solution then to present the flats cellular form, unit surface cell stand density is less relatively.

2, inoblast growing multiplication ability fast

The inoblast of ox, sheep, pig and mouse is cultivated in the 1:3 ratio under the situation about going down to posterity, and conventional nutrient solution then needs 3 days ability confluent culture wares, and uses novel nutrient solution only need get final product the confluent culture ware in 1～1.5 day.Cell to the confluent culture ware is counted demonstration, and the novel nutrient solution cell quantity that uses design is 5～10 times of conventional nutrient solution cell quantity.Prostatropin only adds use once in nutrient solution, the cultured cells that goes down to posterity later on is with respect to only for conventional nutrient solution cultured cells, all having stronger multiplication capacity.The concentration of Prostatropin in nutrient solution surpasses after 4 ng/ml, the equal no significant difference of growthhabit and the influence of the growing multiplication speed of cell and cell.

3, the stronger anti-apoptosis capacity of inoblast

Apoptosis is natural death and the aging phenomenon of cell in process of growth.The inoblast of ox, sheep, pig and mouse is after the novel nutrient solution that uses design; Phenomena of apoptosis seldom occurs when the 5th generation of cultivating; Then the apoptosis phenomenon is very obvious inoblast that conventional nutrient solution is cultivated; Particularly for the inoblast that derives from pig and mouse, using conventional nutrient solution than the novel nutrient solution phenomena of apoptosis of using design increases by 10～50 times.For the inoblast of handling with MTC; Using conventional nutrient solution also to show after 1 week than the novel nutrient solution phenomena of apoptosis of using design increases by 10～50 times, and the novel nutrient solution of display design makes inoblast have stronger anti-apoptosis and antidotal ability as a result.

Above-described embodiment of the present invention does not constitute the qualification to protection domain of the present invention.Any modification of within spirit of the present invention and principle, being done, be equal to replacement and improvement etc., all should be included within the claim protection domain of the present invention.

Claims (1)

1. inoblast nutrient solution is characterized in that: composed of the following components:

10% foetal calf serum, 4 ng/ml Prostatropins, 2.5 μ g/ml Plasmocin, 100 U/ml penicillium mould, 100 μ g/ml Streptomycin sulphates, high sugared DMEM nutrient solution supplies 100%.

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