阅读说明：本技术 一种对间充质细胞标记和示踪方法 (A kind of pair of mesenchymal cell label and tracing method ) 是由 孙桂珍 于 2019-10-15 设计创作，主要内容包括：本发明公开了一种对间充质细胞标记和示踪方法,包括以下方法步骤,S1,将含有目标基因的间充质细胞转入到营养培养基中,诱导细胞进行多次分裂；S2,然后向培养液中加入甘油或二甲亚砜保护剂,置于液氮中,渗透压控制250～325mmol/L；S3,然后切取间充细胞置于100％葡萄糖中10s,充分浸透后膜变为半透明状,然后进行振摇3min；S4,利用荧光染料对其进行充分标记；S5,采用同位素示踪方法进行示踪。本发明的方法采用了多种荧光染料进行标记,整体灵敏度高,方法简便,简化了实验过程,定位定量准确,符合生理条件,同时同位素示踪方法能快速进行定位,并且辐射作用小,准确检测,整个方法较为科学效率,值得推广。(The invention discloses a kind of pair of mesenchymal cell label and tracing methods, comprise the following methods, S1, the mesenchymal cell containing target gene are transferred in nutrient medium, inducing cell is repeatedly divided；Then glycerol or dimethyl sulfoxide protective agent are added into culture solution, is placed in liquid nitrogen by S2, osmotic pressure controls 250~325mmol/L；Then S3 cuts cellula intersitialis and is placed in 10s in 100% glucose, full penetration caudacoria becomes translucent, then carries out shaking 3min；S4 sufficiently marks it using fluorescent dye；S5 carries out tracer using tagging method.Method of the invention uses a variety of fluorescent dyes and is marked, overall sensitivity is high, method is easy, simplifies experimentation, positioning and quantitative is accurate, meet physiological condition, tagging method can be positioned quickly simultaneously, and radiation effects is small, accurate to detect, the more scientific efficiency of entire method, is worthy to be popularized.)

1. a kind of pair of mesenchymal cell label and tracing method, it is characterised in that: comprise the following methods, S1, mesh will be contained The mesenchymal cell of mark gene is transferred in nutrient medium, and inducing cell is repeatedly divided；

Then glycerol or dimethyl sulfoxide protective agent are added into culture solution, is placed in liquid nitrogen by S2, and osmotic pressure control 250~ 325mmol/L；

Then S3 cuts cellula intersitialis and is placed in 10s in 100% glucose, full penetration caudacoria becomes translucent, then carries out Shake 3min；

S4 sufficiently marks it using fluorescent dye；

S5 carries out tracer using tagging method.

2. a kind of pair of mesenchymal cell label according to claim 1 and tracing method, which is characterized in that in step S4, Fluorochrome label method is observed using certain fluorescent dyes that can be integrated on cell membrane or intracellular different tissues, Commonly mainly there are PKH26, DAPI, Dil, CFSE and fluorescin.

3. a kind of pair of mesenchymal cell label according to claim 2 and tracing method, which is characterized in that the fluorescence egg White marker technology is that fluorescence protein gene is imported cell to be marked using genophore, obtains expression fluorescin by screening Cell, can directly be observed by fluorescence microscope, without reaction substrate, fluorescin be can spontaneous generation fluorescence albumen Matter mainly includes green fluorescent protein and its derivative yellow fluorescence protein, blue fluorescent protein and red fluorescent protein； The green fluorescence protein gene have it is efficient, stable, nontoxic, be easy to detect characteristic, can intuitively observe it in living cells Expression can comparatively fast screen the cell of its modification.

4. a kind of pair of mesenchymal cell label according to claim 2 and tracing method, which is characterized in that the carboxyl is glimmering Light element acetoacetate (CFSE) is a kind of fluorescent dye that may pass through cell membrane, into cell after irreversibly with have into the cell The shuttle base fluorescein diacetate group of non-enzymatic hydrolysis effect combines, and is coupled on cell protein, and will not cause cell Apoptosis or death, CFSE occurs cannot release after entering cell from cell, and in conjunction with intracellular protein and Membrane surface proteins, CFSE can be used for viable cell labelling, equably the fluorescence of label is assigned in progeny cell in cell passage, cell warp After CFSE dyeing, during division growth, the CFSE of label can be evenly distributed in progeny cell, make fluorescence in cell point It splits in breeding and is increasingly diluted, fluorescence intensity gradually weakens, and CFSE label is suitable for short-term tracer.

5. a kind of pair of mesenchymal cell label according to claim 2 and tracing method, which is characterized in that the PKH26 A kind of red fluorescence dyestuff, have lipophilicity, can with the lipid domains stable bond of cell membrane, under flag condition, PKH26 does not influence the proliferation and differentiation capability of cell, and when cell division, PKH26 can be equally distributed to 2 progeny cells In, fluorescence intensity is the 1/2 of parental cell, and cell membrane can issue red fluorescence, does not interfere the table of cell membrane surface markers object It reaches, PKH26 can be applied to the inside and outside short-term Cellular tracking of body.

6. a kind of pair of mesenchymal cell label according to claim 1 and tracing method, which is characterized in that in step S5, Tracer method is the micro-analysis method that research object is marked as tracer using radionuclide, using same The radioactive nature of position element carries out autoradiograph to histocyte, micro- sem observation counts, gray scale measures etc., observes cell shape State judges cell function；The nucleic of trace labelling has³H-、¹²⁵I-.³²p-、⁹⁹Tc^m, further include with tritium-labeled deoxidation Thymidine, tritium (³H) deoxythymidine marked is the synthesis precursor of DNA, can be effectively incorporated the S phase In the DNA of cell, tritium can radiate the β ray of low energy, and the influence to cell chromosome is smaller, 7 μm of the range of ray, thin Extracellular substantially radiationless effect, normal tissue damage is small, and ray half-life period 12.3.

Technical field

The present invention relates to medical cell field of engineering technology, especially a kind of pair of mesenchymal cell label and tracing method.

Background technique

Mesenchymal cell is the very low cell of differentiation degree, and core is big, and ovum is oval, and kernel is obvious, and cytoplasm is in weak basophilla.Carefully In cytoplasm in addition to containing a certain number of mitochondrias, there are also a small amount of rough surfaced endoplasmic reticulum (RER), free ribosome, golgiosome and dissipate Lysosome and fat granule etc..The interstitial of mesenchyma is colorless and transparent liquid, removes in cytoplasm and contains a certain number of line grains Except body, there are also a small amount of rough surfaced endoplasmic reticulum (RER), free ribosome, golgiosome and the lysosomes being dispersed in and fat granule etc..Mesenchyma Interstitial be colorless and transparent liquid.There are very strong division and differentiation capability, various knots are not only during embryonic development Form the common ancestor of tissue, and the other kinds of tissue such as differentiation and the endothelium and the smooth muscle that develop into blood vessel.In adult Still some mesenchymal cells with developmental potentiality be can see that in the connective tissue of animal.MSCs is the weight of stem cell line Member is wanted, the Various Tissues cell such as fat, bone, cartilage, cardiac muscle can be divided into, is repairing bone, cartilage and damaged heart cell, It is had great potential in terms for the treatment of inflammation and disease of immune system.However, carrying out clinical treatment using MSCs in the U.S. at present As a result both worried and glad in hundreds of cases: the patient reaction having is good, and some patients do not react then.It understands fully its reason, grinds Study carefully personnel to need to track it after these cells enter human body, sees whether they have been transplanted to suitable position.And it wants Accomplish this point, cell need to be marked using Superparamagnetic Iron Oxide contrast medium, and with mr imaging technique to patient Imaging.

Current traditional labeling method and tracing method to mesenchymal cell, efficiency is lower, takes a long time, and accurate Property it is lower, for above, herein it is proposed that a kind of pair of mesenchymal cell label and tracing method.

Summary of the invention

The present invention provides a kind of pair of mesenchymal cell label and tracing method for the deficiency in background technique.

The present invention is to solve above-mentioned technical deficiency, and using modified technical solution, a kind of pair of mesenchymal cell marks and show Track method, comprises the following methods, S1, and the mesenchymal cell containing target gene is transferred in nutrient medium, induction Cell is repeatedly divided；

Then glycerol or dimethyl sulfoxide protective agent are added into culture solution, is placed in liquid nitrogen by S2, and osmotic pressure control 250~ 325mmol/L；

Then S3 cuts cellula intersitialis and is placed in 10s in 100% glucose, full penetration caudacoria becomes translucent, then Carry out shaking 3min；

S4 sufficiently marks it using fluorescent dye；

S5 carries out tracer using tagging method.

As present invention further optimization mode, in step S4, fluorochrome label method can be combined using certain Fluorescent dye on to cell membrane or intracellular different tissues is observed, and commonly mainly has PKH26, DAPI, Dil, CFSE And fluorescin.

As present invention further optimization mode, the fluorescent protein labeling technology is to utilize genophore by fluorescence egg White channel genes cell to be marked is obtained the cell of expression fluorescin by screening, can directly be observed by fluorescence microscope, Without reaction substrate, fluorescin be can spontaneous generation fluorescence protein, mainly include green fluorescent protein and its derivative Object yellow fluorescence protein, blue fluorescent protein and red fluorescent protein；The green fluorescence protein gene has efficiently, surely It is fixed, nontoxic, be easy to detect characteristic, its expression can be intuitively observed in living cells, can comparatively fast screen its modification cell.

As present invention further optimization mode, the Fluoresceincarboxylic acid acetoacetate (CFSE) is that one kind may pass through carefully The fluorescent dye of after birth, into cell after irreversibly and intracellular two acetic acid of shuttle base fluorescein with non-enzymatic hydrolysis effect Salt groups combine, and are coupled on cell protein, and will not cause apoptosis or death, and CFSE enters after cell just not It can be released from cell, and in conjunction with intracellular protein and Membrane surface proteins, CFSE can be used for viable cell labelling, in cell passage Equably the fluorescence of label is assigned in progeny cell, cell is after CFSE is dyed, during division growth, label CFSE can be evenly distributed in progeny cell, be increasingly diluted fluorescence in cell division breeding, and fluorescence intensity is gradually Weaken, CFSE label is suitable for short-term tracer.

As present invention further optimization mode, the PKH26 is a kind of red fluorescence dyestuff, has lipophilicity, energy Enough and cell membrane lipid domains stable bond, under flag condition, PKH26 does not influence the proliferation and differentiation capability of cell, when When cell division, PKH26 can be equally distributed in 2 progeny cells, and fluorescence intensity is the 1/2 of parental cell, cell membrane Red fluorescence can be issued, does not interfere the expression of cell membrane surface markers object, PKH26 can be applied to the inside and outside short-term cell of body and show Track.

As present invention further optimization mode, in step S5, tracer method be using radionuclide as The micro-analysis method that research object is marked in tracer, radiates histocyte using the radioactive nature of isotope Autography, micro- sem observation count, gray scale measures etc., observe cellular morphology, judge cell function；The core of trace labelling It is known as³H-、¹²⁵I-.³²p-、⁹⁹Tc^m, further include with tritium-labeled deoxythymidine, tritium (³H) the thymidine marked Pyrimidine nucleoside is the synthesis precursor of DNA, can be effectively incorporated in the DNA of S phase cell, and the β that tritium can radiate low energy is penetrated Line, the influence to cell chromosome is smaller, 7 μm of the range of ray, in extracellularly substantially radiationless effect, normal tissue damage It is small, ray half-life period 12.3.

The beneficial effects obtained by the present invention are as follows being: method of the invention uses a variety of fluorescent dyes and is marked, whole High sensitivity, method is easy, simplifies experimentation, positioning and quantitative is accurate, meets physiological condition, while tagging method It can quickly be positioned, and radiation effects is small, accurate to detect, the more scientific efficiency of entire method is worthy to be popularized.

Specific embodiment

Below in conjunction in the embodiment of the present invention, technical solution in the embodiment of the present invention is clearly and completely retouched It states, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based on the present invention In embodiment, every other implementation obtained by those of ordinary skill in the art without making creative efforts Example, shall fall within the protection scope of the present invention.

The present invention provides a kind of technical solution: a kind of pair of mesenchymal cell label and tracing method, including following methods step Suddenly, the mesenchymal cell containing target gene is transferred in nutrient medium by S1, and inducing cell is repeatedly divided；

Then glycerol or dimethyl sulfoxide protective agent are added into culture solution, is placed in liquid nitrogen by S2, and osmotic pressure control 250~ 325mmol/L；

Then S3 cuts cellula intersitialis and is placed in 10s in 100% glucose, full penetration caudacoria becomes translucent, then Carry out shaking 3min；

S4 sufficiently marks it using fluorescent dye；

S5 carries out tracer using tagging method.

In step S4, fluorochrome label method can be integrated on cell membrane or intracellular different tissues using certain Fluorescent dye is observed, and commonly mainly has PKH26, DAPI, Dil, CFSE and fluorescin.

The fluorescent protein labeling technology is that fluorescence protein gene is imported cell to be marked using genophore, passes through sieve Choosing obtains the cell of expression fluorescin, can directly be observed by fluorescence microscope, and without reaction substrate, fluorescin is can The protein of spontaneous generation fluorescence, mainly includes green fluorescent protein and its derivative yellow fluorescence protein, blue fluorescent protein, And red fluorescent protein；The green fluorescence protein gene have it is efficient, stable, nontoxic, be easy to detect characteristic, can be living thin Its expression is intuitively observed in born of the same parents, can comparatively fast screen the cell of its modification.

The Fluoresceincarboxylic acid acetoacetate (CFSE) is a kind of fluorescent dye that may pass through cell membrane, into cell after not Reversibly in conjunction with the intracellular shuttle base fluorescein diacetate group with non-enzymatic hydrolysis effect, it is coupled to cell protein On, and cannot be released from cell after apoptosis or death, CFSE will not be caused to enter cell, and and intracellular protein It is combined with Membrane surface proteins, CFSE can be used for viable cell labelling, and the fluorescence of label is equably assigned to son in cell passage For in cell, cell is after CFSE is dyed, and during division growth, the CFSE of label can be evenly distributed to progeny cell In, it is increasingly diluted fluorescence in cell division breeding, fluorescence intensity gradually weakens, and CFSE label is suitable for short-term Tracer.

The PKH26 is a kind of red fluorescence dyestuff, has lipophilicity, can stablize with the lipid domains of cell membrane and tie It closes, under flag condition, PKH26 does not influence the proliferation and differentiation capability of cell, and when cell division, PKH26 can fifty-fifty divide It is fitted in 2 progeny cells, fluorescence intensity is the 1/2 of parental cell, and cell membrane can issue red fluorescence, not interfere cell membrane The expression of surface marker, PKH26 can be applied to the inside and outside short-term Cellular tracking of body.

In step S5, tracer method be research object is marked using radionuclide as tracer it is micro- Analysis method, using the radioactive nature of isotope carries out autoradiograph to histocyte, micro- sem observation counts, gray scale is surveyed It is fixed etc., cellular morphology is observed, cell function is judged；The nucleic of trace labelling has³H-、¹²⁵I-.³²p-、⁹⁹Tc^m, also wrap It includes with tritium-labeled deoxythymidine, tritium (³H) deoxythymidine marked is the synthesis precursor of DNA, can It is effectively incorporated in the DNA of S phase cell, tritium can radiate the β ray of low energy, and the influence to cell chromosome is smaller, penetrate 7 μm of the range of line, in extracellularly substantially radiationless effect, normal tissue damage is small, and ray half-life period 12.3.

The method of the present invention table is as follows: table 1

Conventional method table is as follows: table 2

It is obvious by Tables 1 and 2, it will be seen that the present invention is more excellent.

To sum up to state, method of the invention uses a variety of fluorescent dyes and is marked, and overall sensitivity is high, and method is easy, Experimentation is simplified, positioning and quantitative is accurate, meet physiological condition, while tagging method can be positioned quickly, and And radiation effects is small, accurate to detect, the more scientific efficiency of entire method is worthy to be popularized.

The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention, for this field skill For art personnel, it is clear that invention is not limited to the details of the above exemplary embodiments, and without departing substantially from spirit of the invention or In the case where essential characteristic, the present invention can be realized in other specific forms.Therefore, in all respects, should all incite somebody to action Embodiment regards exemplary as, and is non-limiting, the scope of the present invention by appended claims rather than on state Bright restriction, it is intended that including all changes that fall within the meaning and scope of the equivalent elements of the claims in the present invention It is interior.

In addition, it should be understood that although this specification is described in terms of embodiments, but not each embodiment is only wrapped Containing an independent technical solution, this description of the specification is merely for the sake of clarity, and those skilled in the art should It considers the specification as a whole, the technical solutions in the various embodiments may also be suitably combined, forms those skilled in the art The other embodiments being understood that.