CN111117955A

CN111117955A - Method for improving breast adipose cell survival rate by using autologous adipose-derived stem cells

Info

Publication number: CN111117955A
Application number: CN202010119002.8A
Authority: CN
Inventors: 白晋
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-02-26
Filing date: 2020-02-26
Publication date: 2020-05-08

Abstract

The invention relates to the field of autologous adipose-derived stem cell application, in particular to a method for improving the survival rate of breast adipose cells by using autologous adipose-derived stem cells, which mainly comprises the steps of extracting autologous fat, extracting and concentrating ADSCs (adsorbent-dependent killer) cells, wherein the extraction of the autologous fat can select a proper part as a supply area and mark the range of extracted fat according to subjective requirements and self conditions of experimenters.

Description

Method for improving breast adipose cell survival rate by using autologous adipose-derived stem cells

Technical Field

The invention relates to the field of autologous adipose-derived stem cell application, in particular to a method for improving the survival rate of breast adipose cells by using autologous adipose-derived stem cells.

Background

The breast is composed of mammary gland and fat, wherein the fat accounts for 90%, the fat has important function for maintaining the elasticity and fullness of the breast, and the fat of the breast is basically the same as the fat of other parts of the body, and the fat of the breast is reduced when the body weight is reduced by physical exercise or weight-reducing medicines. With the continuous improvement of living standard, the number of people requiring breast augmentation is increased year by year, various breast augmentation methods can be selected by plastic doctors and experimenters, and the development and progress of breast augmentation are based on the continuous development and updating of breast augmentation materials. The conventional breast augmentation surgery has several advantages and disadvantages. 1. Injecting the exogenous liquid material to swell the breast: injection of breast augmentation is the earliest way to augment breast, and liquid material is injected into breast tissue or its periphery by injection to achieve the effect of augmenting breast. However, the liquid material can infiltrate into the mammary tissue after being directly injected, and the liquid material cannot be completely taken out when the liquid material is in a problem, and only the mastectomy can be performed, so that the liquid material is applied less and less at present. 2. Self tissue transplantation breast augmentation: in 1895, researchers cut out lipoma of lower limbs to transplant breast-augmentation, initiated the breast-augmentation transplantation of self-tissue, and then followed by the successive emergence of free or pedicel transplantation of composite tissues of fat, dermis, fat and dermal fascia, which are mostly obtained from the abdomen and the upper part of the buttocks, thus facilitating the direct drawing and suturing of the donor area. 3. Augmentation of prosthesis material: the breast augmentation by prosthesis implantation is still the most common method at home and abroad at present. Prostheses are of a wide variety and are classified differently. According to different capsule contents, the capsule can be divided into a silica gel filling type and a normal saline filling type; the method can be divided into injection type and embedded type, wherein the general silica gel filling type is the embedded type, and the physiological saline filling type is the injection type; the surface of the utility model can be divided into a smooth surface and a rough surface; the capsule cavity can be divided into single-capsule type, double-capsule type and multi-capsule type according to the number of the capsule cavities; it can be classified into circular and anatomical forms according to its morphology. 4. Fat particle injection breast augmentation: autologous granular fat injection breast augmentation has received attention from plastic surgeons since the first reports of fat injection augmentation of the breast in 1987. The autologous fat injection has the advantages of no immunological rejection, soft texture of the breast after the operation and the like. With the updating of instruments and the improvement of injection technology, the autologous granular fat is a relatively ideal breast augmentation self-filling material. Compared with the artificial breast augmentation, the autologous fat filling method has the advantages of easily-obtained materials, small complications and sequelae, better operation effect, and more fat from the part with more fat, can achieve the purposes of weight reduction and body shaping while enlarging the breast, and becomes the choice of more and more beauty-loving women. However, the fat necrosis rate of autologous fat filling is relatively high, breast nodules and calcification caused by lipid accumulation are not easy to be identified with other breast diseases, and diagnosis of early breast cancer may be affected, so how to improve the survival rate of fat cells becomes a key factor for improving the success rate of autologous fat filling.

Disclosure of Invention

Aiming at the problems that the fat necrosis rate is relatively high after the prior method for filling and transplanting the human breast by using autologous fat, breast nodules and calcification caused by lipid accumulation are not easy to be identified with other diseases of the breast, and early breast cancer diagnosis can be influenced, the invention provides a method for improving the survival rate of breast fat cells by using autologous fat stem cells, which mainly comprises the steps of extracting autologous fat, extracting and concentrating ADSCs cells, selecting proper parts as supply areas according to the subjective requirements of experimenters and self conditions, and marking the range of extracted fat, wherein the extraction and concentration of the ADSCs cells are mainly divided into the following steps:

step one, repeatedly washing the extracted adipose tissues by sterile PBS (phosphate buffer solution) until the adipose tissues are free of blood color;

step two, subpackaging the tissue liquid in the step one into a centrifuge tube, carrying out centrifugal operation by using a freezing centrifuge, wherein the centrifuged tissue is divided into four layers in the centrifuge tube from top to bottom, wherein the four layers are sequentially divided into a grease layer, a fat layer, a physiological saline layer and a cell precipitation layer, the grease layer is discarded, and then the fat layer is collected into the centrifuge tube again;

step three, weighing collagenase by using an electronic balance, adding the collagenase into normal saline, stirring to fully dissolve the collagenase, extracting a collagenase solution by using a sterile syringe, sterilizing by using a filter, and mixing the collagenase solution according to the proportion of 1: 1, adding a collagenase solution into a centrifugal tube, fully oscillating the centrifugal tube to uniformly mix collagenase and fat, and putting the centrifugal tube into a water bath box or an incubator for digestion;

step four, digesting the fat in the centrifuge tube in the previous step into sticky paste, centrifuging by using a freezing centrifuge, dividing the tissue in the centrifuge tube into three layers from top to bottom after centrifugation, namely a fat layer, a physiological saline layer and a precipitation layer in sequence, removing the upper two layers, taking the precipitation layer in each centrifuge tube, and placing the precipitation layers in the same centrifuge tube;

fifthly, adding physiological saline into the centrifugal tube in the previous step, filtering by using a filter screen, and then putting the centrifugal tube into a refrigerated centrifuge for centrifugal operation, wherein the last layer of precipitated cells in the centrifugal tube are remained to be ADSCs for transplantation;

step six, inoculating the precipitated cells in the step one into a culture dish, placing the culture dish into an incubator for incubation, changing the liquid the next day, observing every day, and changing the liquid once every 3 days;

the extraction of the autologous fat comprises the following specific operations: performing swelling anesthesia in a conventional supply area, preparing local anesthesia swelling liquid by using 500mL of 0.9% physiological saline, 40mL of 2% lidocaine hydrochloride and 1mL of epinephrine, making a 3-5mm skin incision on a marked suction part, placing one hand on the surface of the skin of the suction part, sensing the positioning and swelling degree of a needle, uniformly injecting the liquid in the range of the marked supply area until the swelling is whitish and the skin has an orange peel-like appearance, lightly massaging an operation area until the swelling liquid is uniformly diffused after the injection is finished, inserting subcutaneous adipose tissue along the incision after the anesthesia is successful, and uniformly extracting deep fat in a sector shape by using a liposuction needle until enough amount of fat is obtained;

further, in the first step, the sterile PBS solution is washed to be 0.1M PBS, pH7.2-7.4, and the proportioning method is as follows: 1000ml of distilled water was added with NaCI 9g and Na₂HPO₄·2H ₂0 6g、NaH₂PO·2H ₂0 0.4g；

Further, in the second step, the tissue liquid in the previous step is subpackaged into 50ml centrifuge tubes, and centrifuged for 5min at 1200 r/min by using a freezing centrifuge;

further, in the third step, collagenase type I is weighed with an electronic balance with an accuracy of 0.001 g, added to normal saline, stirred to sufficiently dissolve collagenase to a concentration of 0.1%, the collagenase solution is extracted with a sterile syringe, sterilized through a 0.22 μm filter, and mixed in a ratio of 1: adding collagenase solution at a ratio of 1, fully oscillating the centrifuge tube to mix collagenase and fat, and placing in a 37 deg.C water bath tank or 5% CO₂Digesting in an incubator for 45min, taking out every 10min, and shaking up;

further, in the fourth step, the fat is digested into paste, and the paste is centrifuged for 5min at 1200 r/min by a freezing centrifuge;

further, in the fifth step, the physiological saline is added into the centrifugal tube in the previous step, the mixture is filtered by a 70-micron filter screen and then is placed into a refrigerated centrifuge for centrifugation at 1200 r/min for 5 min; leaving the last layer of settled cells in the centrifuge tube;

further, the fifth step needs to be repeated for three times;

further, the method comprisesIn the sixth step, the precipitated cells are inoculated into a culture dish and placed at 37 ℃ and 5% CO₂Incubate in the incubator, change the solution the next day, observe every day later, change the solution once every 3 days.

The invention has the beneficial effects that: the invention provides a method for improving the survival rate of breast fat cells by using autologous fat stem cells, which can greatly reduce the fat necrosis rate after the filling transplantation of the breast of a human body by using autologous fat, improve the survival rate of female breast transplanted fat cells, and reduce the morbidity of complications and sequelae thereof. The method has the advantages of good fat survival effect, fewer complications, simple process, capability of meeting the use requirements of people and great application value.

Drawings

FIG. 1 is a graph of fat gain after nude mouse transplantation;

FIG. 2 is a graph showing comparative analysis of fat acquisition after nude mouse transplantation;

FIG. 3 is a table comparing clinical data of two groups of experimenters;

FIG. 4 is a chart of two groups of experimenters satisfaction questionnaires;

FIG. 5 is a statistical table of post-operative chest circumference maintenance of experimenters;

fig. 6 shows the values of post-operative chest elevations and their retention in the experimenter.

Detailed Description

It should be noted that the present invention is directed to a method for improving the survival rate of adipose cells in the breast by using autologous adipose-derived stem cells for non-therapeutic purposes, which is not a diagnostic and therapeutic method for human body all the time, and the technical solution of the present invention will be clearly and completely described with reference to the following embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. It should be noted that: in the present invention, all the embodiments and preferred methods mentioned herein can be combined with each other to form a new technical solution, if not specifically stated. In the present invention, all the technical features mentioned herein and preferred features may be combined with each other to form a new technical solution, if not specifically stated. In the present invention, the components referred to or the preferred components thereof may be combined with each other to form a novel embodiment, if not specifically stated. In the present invention, unless otherwise stated, the numerical range "a-b" represents a shorthand representation of any combination of real numbers between a and b, where a and b are both real numbers. For example, a numerical range of "1.5 to 2.5" means that all real numbers between "1.5 to 2.5" have been listed herein, and "1.5 to 2.5" is only a shorthand representation of the combination of these values. The "ranges" disclosed herein may have one or more lower limits and one or more upper limits, respectively, in the form of lower limits and upper limits. In the present invention, unless otherwise specified, the individual reactions or operation steps may be performed sequentially or may be performed in sequence. Preferably, the reaction processes herein are carried out sequentially. Unless otherwise defined, technical and scientific terms used herein have the same meaning as is familiar to those skilled in the art. In addition, any methods or materials similar or equivalent to those described herein can also be used in the present invention.

Specific example 1: in order to explore whether the invention is really effective, a BALB/c nude mouse animal model test is carried out, and the specific implementation steps are as follows:

obtaining adipocytes

1.1 extraction of fat: according to subjective requirements and self conditions of experimenters, selecting proper parts as supply regions and marking the range of extracted fat, the specific operation is as follows: the general supply area swelling anesthesia is characterized in that 0.9% physiological saline 500mL, 2% lidocaine hydrochloride 40mL and epinephrine 1mL are prepared into a local anesthesia swelling solution, a 3-5mm skin incision is made at a marked suction position, one hand is placed on the surface of the skin at the suction position, the positioning and swelling degree of a needle can be sensed, and the injection is uniformly carried out in the range of the marked supply area until swelling becomes white and the skin has an orange peel appearance. After the injection is finished, the operated area should be massaged lightly until the swelling solution is spread evenly. After the anesthesia succeeds, inserting subcutaneous adipose tissues along the incision by using a liposuction needle, and uniformly extracting deep fat in a fan shape to a sufficient amount by using the liposuction needle;

extracting and concentrating ADSCs and fat cells: the process is divided into 6 steps, and the specific operation is as follows:

(1) adding NaCI 9g and Na into the obtained subcutaneous adipose tissue with PBS (0.1M PBS, pH7.2-7.4, preparation method: 1000ml distilled water)₂HPO₄·2H ₂0 6g、NaH₂PO·2H₂00.4 g) repeatedly washing adipose tissue until there is no blood color;

(2) subpackaging the tissue liquid obtained in the previous step into 50ml centrifuge tubes, centrifuging for 5min at 1200 r/min by using a centrifuge, dividing the tissue into four layers in the centrifuge tube after centrifuging, sequentially dividing the tissue into a grease layer, a fat layer, a physiological saline layer and a cell precipitation layer from top to bottom, discarding the grease layer, and collecting the fat layer into the centrifuge tubes again;

(3) type I collagenase (product of Gibco) was weighed with an electronic balance (accuracy 0.001 g) and stirred with physiological saline to sufficiently dissolve collagenase to prepare a concentration of 0.1%, and the collagenase solution was extracted with a sterile syringe, sterilized through a 0.22 μm filter (Biofil, product of TaKaRa), and mixed in a centrifuge tube in the above step at a ratio of 1: adding collagenase solution at a ratio of 1, fully oscillating the centrifuge tube to mix collagenase and fat, and placing in a 37 deg.C water bath tank or 5% CO₂Digesting in an incubator for 45min, taking out every 10min, and shaking up;

(4) digesting the fat in the centrifuge tube in the previous step into paste, centrifuging for 10min at 1200 r/min by a centrifuge, dividing the centrifuged tissue into three layers from top to bottom in the centrifuge tube, namely a fat layer, a physiological saline layer and a precipitation layer in sequence, removing the upper two layers, taking the precipitation layer in each centrifuge tube, and placing the precipitation layers in the same centrifuge tube;

(5) adding physiological saline to 20ml in the centrifuge tube in the previous step, filtering with a 70 u m filter screen, centrifuging for 5min at 1200 r/min by a centrifuge, and leaving the lowest layer of precipitated cells in the centrifuge tube as ADSCs (adipose-derived stem cells) (repeating the step for 3 times);

(6) finally, the precipitated cells obtained in the above step were inoculated into a culture dish and placed at 37 ℃ in 5% CO₂Incubating in the incubator, changing the liquid the next day, observing every day, and changing the liquid once every 3 days;

BALB/c nude mouse animal model test

2.1 preparation of the graft

Mixing the adipose-derived stem cells extracted according to the steps with autologous adipose cells in proportion, and then naming the mixture as a research group, naming the mixture as a control group according to the traditional adipose cell transplantation method, and naming the mixture as a blank group after mixing the adipose cells with normal saline;

2.2 BALB/c nude mouse animal model test

Firstly, 10 BALB/c nude mice of 6 weeks old are taken and divided into (A, B and C) 3 groups, 5 mice in each group are injected with 1% sodium pentobarbital in tail vein, and fat graft injection is performed subcutaneously on the two sides of the back of the nude mice after the nude mice are completely anesthetized. Injecting 1ml and 2ml respectively at both sides of the back of each nude mouse by using a fan-shaped multi-point injection mode, starting from a central point, dividing into 8 directions, injecting by using a needle withdrawing method with the needle inserting distance of 1cm, and injecting 0.125ml in each direction. Injecting a study group fat graft into a nude mouse of the group A, injecting a control group fat graft into the group B, and injecting a blank group fat graft into the group C;

2.3 fat acquisition and weighing and analysis after nude mouse transplantation

After 12 weeks of fat transplantation, all nude mice are subjected to intraperitoneal injection of 400uL of 1% sodium pentobarbital and killed, then each nude mouse is dissected to obtain all fat tissues of two transplantation parts, weighing and recording are carried out, the recorded result is shown in the specification and figure 1, the fat weight average values of three groups are compared and analyzed, the result is shown in the specification and figure 2, the fat weight average value of the group A is 1.59g, the fat weight average value of the group B and the fat weight average value of the group C are 1.03g and 0.46g respectively, after 12 weeks, the weight of the remaining fat tissues of the group A is obviously higher than that of the group B and the group C, the survival rate of fat cells after fat transplantation can be effectively improved by adopting fat stem cells to assist the fat particle transplantation, and the experimental result also shows that the success rate of the fat transplantation can be improved by using the fat stem cells to assist the.

Example 2: in order to solve the problem that a certain degree of absorption rate exists after autologous fat transplantation, the invention is illustrated by the following experiment, and the specific implementation steps are as follows:

acquisition of autologous ADSCs and adipocytes

1.1 extraction of autologous fat: according to subjective requirements and self conditions of experimenters, the upper arm, the abdomen, the inner side and the outer side of the thigh are selected as supply areas and the range of extracted fat is marked. The specific operation is as follows: the local anesthesia swelling liquid is prepared by 500mL of 0.9% normal saline, 40mL of 2% lidocaine hydrochloride and 1mL of epinephrine in a conventional region swelling anesthesia manner, a 3-5mm skin incision is made at a marked suction position, one hand is placed on the surface of the skin at the suction position, the positioning and swelling degree of a needle can be sensed, and the injection is uniformly carried out in the range of the marked supply region until swelling becomes white and the skin has an orange peel appearance. After the injection is finished, the operated area should be massaged lightly until the swelling solution is spread evenly. After the anesthesia is successful, inserting subcutaneous adipose tissues along the incision by using a liposuction needle, and uniformly extracting about 500mL of deep fat in a fan shape by using the liposuction needle;

1.2 extraction and concentration of ADSCs and fat cells: the process is divided into 6 steps, and the specific operation is as follows:

(3) weighing type I collagenase (Gibco product) with an electronic balance (accuracy of 0.001 g), adding normal saline, stirring, and dissolving collagenase to obtain the final product0.1% strength, collagenase solution was drawn off with a sterile syringe, sterilized through a 0.22 μm filter (Biofil, product TaKaRa) and introduced into the centrifuge tube in the preceding step at a rate of 1: adding collagenase solution at a ratio of 1, fully oscillating the centrifuge tube to mix collagenase and fat, and placing in a 37 deg.C water bath tank or 5% CO₂Digesting in an incubator for 45min, taking out every 10min, and shaking up;

1.3 fat cell extraction: about 500mL of adipose tissue was extracted from each experimenter, 100 mL of which was used to extract ADSCs according to the above procedure, and the remaining about 400 mL of adipose tissue was used as filling fat, which was cryo-preserved on crushed ice and treated as follows:

① placing the sucked fat liquid and the injector into 4 deg.C physiological saline, standing for 5min, dividing the mixed solution into 3 layers, the upper layer is orange fat liquid, the middle layer is light yellow fat particles, and the lower layer is pink swelling liquid and blood mixture;

② taking out and draining the bottom liquid, transferring the middle and upper layers of fat liquid and fat particles into a 50ml centrifuge tube, placing into a refrigerated centrifuge, centrifuging at 400 r/min for 2min to obtain upper and lower layers, wherein the upper layer is light yellow fat and the bottom layer is light red swelling liquid, and removing the lower layer of liquid to obtain the transplanted adipocytes.

Second, the back transfusion of fat cell (transplantation of autologous fat-derived stem cell)

Routine chest disinfection is started before, a proper needle insertion point is selected at a position 2cm away from the marked edge of the receiving area, and local infiltration anesthesia is performed by lidocaine. Before chest injection, an incision is made with a percutaneous needle over the outside of the breast. Fat was injected using an injection needle 2mm in diameter and 20 cm in length and a 10 mL syringe. Touching the position and the depth of the needle head with the left hand, injecting with the right hand, and finally injecting 150-250 mL of fat into each mammary gland. Injection sites were 3: injecting 50% of the total amount below the pectoralis major, then injecting 25% above the pectoralis major, and finally injecting the remaining 25% in the breast tissue and subcutaneous fat layer, and after the injection is completed, gently massaging and shaping and caring with a bandage. The step is divided into two groups, the research group is that the ADSCs and fat cell mixture obtained according to the steps are processed, then the purified fat particle tissue is fully mixed with the obtained ADSCs, the mixture is kept still for 30 min, the ADSCs and the fat particle cells are fully attached to form the ADSCs assisted fat particle transplantation mixture, the mixture is returned to the chest of an experimenter, the contrast group is carried out according to the conventional fat particle transplantation mixture method, the number of people in the research group is n =46, and the number of people in the contrast group is n = 33.

Third, evaluation of Effect

3.1 setting quantitative index, calculating postoperative scores of two groups of experimenters according to the scoring standard, and comparing whether the two groups have difference in treatment effect. Observation indexes and judgment standards: two groups of subjects were compared for 12 months post-surgery scoring. The standard is as follows: postoperative scar condition, postoperative complication condition, breast augmentation effect, and experimenter satisfaction. Respectively representing 4 grades by 0-3 points, adding all the grades to obtain a final score, wherein the total score is 0-12 points, and the higher the score is, the better the operation effect is;

3.1.1 postoperative scar Scoring Standard

0 minute, obvious scar exists after operation, or scar hyperplasia condition appears

1 minute, obvious scar exists after the operation, and scar hyperplasia does not occur

2 minutes, no obvious scar exists after the operation, and no scar hyperplasia occurs

3 min, no scar is left after the operation, or the scar is completely healed and disappeared

3.1.2 postoperative complication Scoring Standard

0 point, infection, necrosis, granuloma or other serious symptoms after operation

1 minute, subcutaneous hemorrhage after operation and self-recovery after operation

2 points, swelling and other symptoms appear after the operation and the postoperative recovery is self-performed

3 minutes, no complication appeared after the operation

3.1.3 postoperative Breast augmentation score Standard (12 months after operation for Observation)

0min, no breast enlargement after operation

1 minute, the breast after operation is increased by less than 0.5 cup

2 minutes, increase 0.5~1 cup of breast after the operation

3 min, adding more than 1 cup for breast after operation

3.1.4 experimenter satisfaction scoring criteria

Score 0, the experimenter is not satisfied with the effect of the operation

1 minute, the experimenter feels the effect of the operation

2 points, the experimenter is satisfied with the operation effect

3 points, the experimenter is very satisfied with the operation effect

3.2 evaluating the bulging degree of the breasts, and comparing the chest circumference and the chest bulging value of the two groups and the stable retention rate of the two groups. By bust is meant the maximum circumference through the exterior of the breast. The breast eminence value refers to the vertical distance from the root of the nipple to the plane of the sternum. During measurement, the experimenter takes a natural end sitting position, the shoulders are swung flat, the arms are naturally drooped, the chest is straight, the eyes are gazed ahead, the patient breathes naturally, the measurer takes a flexible ruler to measure the chest circumference and the breast bulge value, the chest circumference and the breast bulge value are measured for 2 times respectively, and the average value is taken. Data analysis was performed using SPSS 20.0 statistical software. The measured data are expressed by mean plus or minus standard deviation, and variance analysis designed by repeated measurement is compared; the count data are expressed in terms of rate (%) and the comparison is X²Inspection, P<A difference of 0.05 is statistically significant.

Fourth, effect verification

4.1 comparison of clinical data of two groups of experimenters

As shown in the attached figure 3 of the specification, the data of age, BMI, follow-up time, supply area and the like have no obvious difference and do not have statistical significance, the chest injection amount of a control group is higher than that of a research group (P < 0.001), wherein the fat cell absorption phenomenon appears in the traditional ADSCs assisted fat transplantation group within 5-6 months after the operation, and the research group still maintains a satisfactory effect within 6-13 months after the operation. The difference between the two surgeries required by some experimenters at 6-9 months after surgery, the two surgeries performed by 8 cases (17.4%) in the study group and 11 cases (33.3%) in the control group was statistically significant (P = 0.011). All subjects operated successfully without infection, necrosis, granuloma or other serious complications. In the control group, 23 subjects developed a symptom of swelling of the chest after surgery, and recovered for about 2 weeks. In the study group, 34 experimenters have slight swelling on the chest, recover 1-2 weeks after operation and have no subcutaneous bleeding;

4.2 set the quantified index score comparison

The postoperative scores of the two groups of experimenters were calculated according to the scoring criteria, and the two groups were compared for differences in therapeutic effect. Observation indexes and judgment standards: two groups of subjects were compared for 12 months post-surgery scoring. The standard is as follows: postoperative scar condition, postoperative complication condition, breast augmentation effect, and experimenter satisfaction. 0-3 points are respectively adopted to represent 4 grades, the final score is obtained by adding all the scores, the total score is 0-12 points, and the higher the score is, the better the operation effect is. The final score of the method for assisting adipocyte transplantation by using the adipose-derived stem cells is far higher than that of the common adipose-derived transplantation, so that the method for assisting adipocyte transplantation by using the adipose-derived stem cells can enhance the shaping effect and improve the satisfaction of experimenters, and the specific situation is shown in the attached figure 4 of the specification;

4.3 evaluating the degree of breast bulging, comparing the bust circumference, chest bulging value and stable retention rate

The measured bust refers to the maximum circumference through the exterior of the breast. The breast eminence value refers to the vertical distance from the root of the nipple to the plane of the sternum. During measurement, the experimenter takes a natural end sitting position, the shoulders are swung flat, the arms are naturally drooped, the chest is straight, the eyes are gazed ahead, the patient breathes naturally, the measurer takes a flexible ruler to measure the chest circumference and the breast bulge value, the chest circumference and the breast bulge value are measured for 2 times respectively, and the average value is taken. The difference between the chest circumference before operation of two groups of experimenters is not statistically significant (P is more than 0.05). Comparing the chest circumferences of two groups of patients 2 weeks after operation and 1, 3 and 6 months, and adopting variance analysis of repeated measurement design, wherein the result shows that the chest circumferences of the experimenters at different time points after the operation are different, and the chest circumference and chest rising value of each time point after the operation of the research group are higher than those of the control group; and the stable retention rate of the chest circumference and the chest bulge value at each time point after the operation of the study group is higher than that of the control group, and the specific situation is shown in the specification, the attached figure 5 and the specification, the attached figure 6. In conclusion, compared with the traditional adipose-derived stem cell assisted adipose cell transplantation method, the survival rate of the breast transplanted adipose cells is obviously improved by the method of the ADSCs assisted adipose transplantation technology, the satisfaction of experimenters after breast filling is effectively improved, and the morbidity of complications and sequelae of the experimenters is reduced.

Claims

1. A method for improving the survival rate of breast adipose cells by using autologous adipose-derived stem cells is characterized by mainly comprising the steps of extracting autologous fat, extracting and concentrating ADSCs (adipose derived stromal cells), selecting a proper part as a supply area and marking the range of extracted fat according to subjective requirements and self conditions of experimenters in the extraction of the autologous fat, wherein the extraction and concentration of the ADSCs are mainly divided into the following steps:

and step six, inoculating the precipitated cells in the step one into a culture dish, placing the culture dish into an incubator for incubation, changing the liquid the next day, observing every day, and changing the liquid every 3 days.

2. The method for improving the survival rate of the adipose cells of the breast by using the autologous adipose-derived stem cells according to claim 1, wherein the specific operations of extracting the autologous fat are as follows: the method comprises the steps of performing swelling anesthesia in a conventional supply area, preparing local anesthesia swelling liquid from 500mL of 0.9% physiological saline, 40mL of 2% lidocaine hydrochloride and 1mL of epinephrine, making a 3-5mm skin incision at a marked suction position, placing one hand on the surface of the skin at the suction position, enabling the needle to be positioned and swollen, uniformly injecting the liquid in the range of the marked supply area until swelling becomes white, enabling the skin to have an orange peel-like appearance, lightly massaging an operation area until the swelling liquid is uniformly diffused after injection is finished, inserting subcutaneous adipose tissues along the incision after anesthesia is successful, and uniformly extracting deep fat in a fan shape until enough amount.

3. The method for improving the survival rate of the adipose cells of the breast by using the autologous adipose-derived stem cells, according to claim 1, is characterized in that, further, in the step one, the sterile PBS solution is used for washing to be 0.1M PBS, pH is 7.2-7.4, and the ratio isThe method comprises the following steps: 1000ml of distilled water was added with NaCI 9g and Na₂HPO₄·2H₂0 6g、NaH₂PO·2H₂0 0.4g。

4. The method for improving the survival rate of the adipose cells of the breast by using the autologous adipose-derived stem cells, according to the claim 1, is characterized in that, in the second step, the tissue fluid in the previous step is subpackaged into 50ml centrifuge tubes, and the centrifuge tubes are centrifuged for 5min at 1200 r/min by using a refrigerated centrifuge.

5. The method for improving the survival rate of the adipose cells of the chest by utilizing the autologous adipose-derived stem cells, according to the claim 1, is further characterized in that, in the third step, the collagenase type I is weighed by an electronic balance with the accuracy of 0.001 g and is added into the normal saline to be stirred, so that the collagenase is fully dissolved and is configured to be 0.1% in concentration, the collagenase solution is extracted by a sterile syringe, and is sterilized by a 0.22 μm filter according to the proportion of 1: adding collagenase solution at a ratio of 1, fully oscillating the centrifuge tube to mix collagenase and fat, and placing in a 37 deg.C water bath tank or 5% CO₂Digesting in incubator for 45min, taking out every 10min, and shaking up.

6. The method for improving the survival rate of the adipose cells of the breast by using the autologous adipose tissue stem cells, according to the claim 1, is characterized in that in the fourth step, the adipose tissues are further digested into a sticky paste, and the sticky paste is centrifuged for 5min at 1200 r/min by using a refrigerated centrifuge.

7. The method for improving the survival rate of the adipose cells of the breast by utilizing the autologous adipose-derived stem cells is characterized in that, further, in the fifth step, the physiological saline is added into the centrifugal tube in the previous step, the mixture is filtered by a 70-micron filter screen, and then the mixture is placed into a refrigerated centrifuge for centrifugation at 1200 r/min for 5 min; the last layer of pelleted cells in the centrifuge tube was left behind.

8. The method for improving the survival rate of the adipose cells of the breast by utilizing the autologous adipose-derived stem cells is characterized in that the fifth step is further repeated three times.

9. The method for improving the survival rate of the adipose cells of the breast by utilizing the autologous adipose-derived stem cells, according to the claim 1, is characterized in that, further, in the sixth step, the precipitated cells are inoculated into a culture dish and are placed at 37 ℃ and 5% CO₂Incubate in the incubator, change the solution the next day, observe every day later, change the solution once every 3 days.