CN110172439A - A kind of extraction of islet cells and enrichment procedure - Google Patents
A kind of extraction of islet cells and enrichment procedure Download PDFInfo
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- CN110172439A CN110172439A CN201910334359.5A CN201910334359A CN110172439A CN 110172439 A CN110172439 A CN 110172439A CN 201910334359 A CN201910334359 A CN 201910334359A CN 110172439 A CN110172439 A CN 110172439A
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- C12N5/0676—Pancreatic cells
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Abstract
The present invention relates to cell engineering fields, extraction and enrichment procedure more particularly to a kind of islet cells, the extraction of the islet cells realizes that the cleaning solution includes by a kind of cell tissue cleaning solution: Hank ' s liquid, apple extract, glycyrrhiza glabra root extract；The culture proliferation of the islet cells is realized by a kind of islet cell culture base, the islet cell culture base includes: RPMI-1640 culture medium, DMEM/F12 culture medium, fetal calf serum, the 3- hydroxyl xylose glucose of phloretin -2 ' glycosides, mycillin solution (penicillin 10000U/ml, streptomysin 10000ug/ml), apple extract, Gotu Kola P.E；Islet cells of the present invention extracts and culture enrichment procedure, islet B cell proliferation can be effectively facilitated, the extraction of the islet cells and enrichment procedure are remarkably improved the quantity and activity of islet cells obtained, can be used for internal cell transplantation for diabetes, and potential applicability in clinical practice is good.
Description
Technical field
The present invention relates to cell engineering fields, and in particular to a kind of extraction of islet cells and enrichment procedure.
Background technique
Glucagon is a kind of proteohormone by islet cells secrete, it plays a role to adjust together with insulin
Save the level of blood glucose.When blood glucose (glucose) concentration is low, glucagon stimulates liver to convert grape for the glycogen of storage
Sugar, and be immediately released into blood flow, the concentration of blood glucose is increased with this.It is raw that glucagon can also increase protein in liver
At the rate of glucose.
Pancreas is then the important organ of animal body, is made of endocrine gland and exocrine gland tissue.Endocrine gland tissue
As our usually said islet cells, are dispersed in and are embedded in exocrine gland tissue.Islet cells is by α, β and delta cell group
At cell mass not of uniform size, wherein β cell is the exclusive source of insulin secretion, the missing meeting of its quantity and function
Cause internal body blood sugar concentration abnormal, so as to cause diabetes.Therefore, islet cells is the important of diabetes molecular pathology
Research object, while islet cell transplantation is the unique treatment means of Late-stage diabetic people.
The molecular pathology research of diabetes and the transplantation experiments of islet cells require lot of pure, be not mingled with outer point
Secrete the islet cells of glandular tissue.In order to achieve this goal, researcher continuously improves the method isolated and purified, and makes every effort to provide
Standardized reagent, especially for digesting the enzymatic reagent of body of gland.However, traditional collagenase dissociation is lacked there is several
It falls into, such as the tissue of separate sources or the enzyme of different batches, the reproducibility of enzyme effect is poor.In addition, clinical at present
The pancreas islet source of acquisition is very limited, needs that a small amount of pancreas islet of donor source is sufficiently extended and is proliferated in vitro.However, at present
The cell culture medium of the common culture islet cells used, it is difficult to which quickly and effectively amplification obtains a large amount of islet cells.
Summary of the invention
It is an object of the invention to propose that a kind of simple, safe and effective islet cells extracts and cultivate enrichment procedure, institute
The extraction and enrichment procedure of stating islet cells are remarkably improved the quantity of islet cells obtained.
Above-mentioned technical problem to be solved by this invention, is achieved by following technical solution:
A kind of extraction of islet cells and enrichment procedure, the culture proliferation side of extraction step and islet cells comprising islet cells
Method.
As a preferred embodiment, the extraction of the islet cells is realized by a kind of cell tissue cleaning solution, described
Cleaning solution includes: Hank ' s liquid, apple extract, glycyrrhiza glabra root extract；The content of the apple extract is Hank ' s
The 5 ~ 9% of liquid quality；The content of the glycyrrhiza glabra root extract is the 3 ~ 6% of Hank ' s liquid quality.
As a preferred embodiment, the culture proliferation of the islet cells is realized by a kind of islet cell culture base, institute
The islet cell culture base stated includes: RPMI-1640 culture medium, DMEM/F12 culture medium, fetal calf serum, 3- hydroxyl phloretin-
2 ' xylose glucose glycosides, mycillin solution (penicillin 10000U/ml, streptomysin 10000ug/ml), apple extract, accumulated snow
Careless extract.
As a preferred embodiment, the content of the DMEM/F12 culture medium be RPMI-1640 culture medium quality 30 ~
45%；The content of fetal calf serum is the 6 ~ 10% of RPMI-1640 culture medium quality；The 3- hydroxyl xylose glucose glycosides of phloretin -2 '
Content is the 0.5 ~ 1% of RPMI-1640 culture medium quality；Mycillin solution (penicillin 10000U/ml, streptomysin 10000ug/
Ml content) is the 0.4 ~ 0.9% of RPMI-1640 culture medium quality；The content of apple extract is RPMI-1640 culture substrate
The 2 ~ 4.5% of amount；The content of Gotu Kola P.E is the 1 ~ 2.5% of RPMI-1640 culture medium quality.
As a preferred embodiment, the apple extract is prepared by the inclusion of the method for following steps: by apple
It is smashed to pieces after fruit cleaning, the ethanol water that volume fraction is 60 ~ 80% is added with solid-liquid ratio 1:1.5 ~ 2, it is close with 350 ~ 450W of power
It seals and is ultrasonically treated 50 ~ 80min, by filtrate rotary evaporation after suction filtration, then obtain the apple extract after macroreticular resin separates.
As a preferred embodiment, the Gotu Kola P.E is prepared by the inclusion of the method for following steps: will accumulate
Snow grass is ground after drying sterilization, addition acidic alcohol solvent, 50 ~ 60min of ultrasound at 60 ~ 65 DEG C, after filtering, drying
The Gotu Kola P.E.
As a preferred embodiment, the glycyrrhiza glabra root extract is prepared by the inclusion of the method for following steps
To: it is ground after glycyrrhiza glabra root is dried sterilization, acidic alcohol solvent, 80 ~ 90min of ultrasound, mistake at 70 ~ 75 DEG C is added
The glycyrrhiza glabra root extract is obtained after filter, drying.
As a preferred embodiment, the extraction of islet cells is realized by the inclusion of the method for following steps: choosing just birth
The healthy mice pancreas of non-lactation is placed in 5 in the cell tissue cleaning solution at 3 ~ 6 DEG C after removing coating, blood vessel and connective tissue
Then ~ 7h is that pancreas islet is separated with its hetero-organization, then obtains pancreas islet after glucan is interrupted density gradient centrifugation by mechanical lapping
Cell.
As a preferred embodiment, the culture enrichment procedure of islet cells is prepared by the inclusion of the method for following steps
To: obtained islet cells will be extracted by 1 × 105The density of a/ml is inoculated in the culture equipped with the islet cell culture base
It in ware, is cultivated at 35 ~ 38 DEG C of constant temperature 4 ~ 10 days, then replaces the continuation of islet cell culture base and cultivated at 35 ~ 38 DEG C of constant temperature,
Islet cells after being proliferated.
As a preferred embodiment, the islet cells is islet B cell.
The utility model has the advantages that islet cells of the present invention extracts and culture enrichment procedure, by using in breeding
Islet cell culture base of the present invention can effectively facilitate islet B cell proliferation, the extraction and proliferation of the islet cells
Method is remarkably improved the quantity and activity of islet cells obtained, can be used for internal cell transplantation for diabetes, clinical application
It has good prospects.
Specific embodiment
It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention
Technical solution be clearly and completely described, it is clear that described embodiments are some of the embodiments of the present invention, rather than
Whole embodiments.Based on the embodiments of the present invention, those of ordinary skill in the art are not making creative work premise
Under every other embodiment obtained, shall fall within the protection scope of the present invention.
The extraction of 1 islet B cell of embodiment and culture enrichment procedure
The extracting method of islet B cell:
Cleaning solution includes: Hank ' s liquid, apple extract, glycyrrhiza glabra root extract；The content of the apple extract is
The 8% of Hank ' s liquid quality；The content of the glycyrrhiza glabra root extract is the 4.5% of Hank ' s liquid quality.
The healthy mice pancreas for choosing the just non-lactation of birth is placed at 4 DEG C after removing coating, blood vessel and connective tissue
Then 6 h in cell tissue cleaning solution are that pancreas islet is separated with its hetero-organization, then is interrupted density level bands through glucan by mechanical lapping
Islet cells is obtained after degree centrifugation.
The culture enrichment procedure of islet B cell:
Islet cell culture base includes: RPMI-1640 culture medium, DMEM/F12 culture medium, fetal calf serum, 3- hydroxyl phloretin-
2 ' xylose glucose glycosides, mycillin solution (penicillin 10000U/ml, streptomysin 10000ug/ml), apple extract, accumulated snow
Careless extract；The content of the DMEM/F12 culture medium is the 37% of RPMI-1640 culture medium quality；The content of fetal calf serum is
The 8% of RPMI-1640 culture medium quality；The content of the 3- hydroxyl xylose glucose glycosides of phloretin -2 ' is RPMI-1640 culture substrate
The 0.7% of amount；The content of mycillin solution (penicillin 10000U/ml, streptomysin 10000ug/ml) is RPMI-1640 culture
The 0.7% of matrix amount；The content of apple extract is the 3.5% of RPMI-1640 culture medium quality；The content of Gotu Kola P.E is
The 2% of RPMI-1640 culture medium quality；
The method of the apple extract as follows is prepared: smashing to pieces after apple is cleaned, with solid-liquid ratio 1:1.8
The ethanol water that volume fraction is 70% is added, 65 min of ultrasonic treatment are sealed with power 400W, filtrate is rotated after suction filtration and is steamed
Hair, then the apple extract is obtained after macroreticular resin separates；
The method of the Gotu Kola P.E as follows is prepared: grinding, adds after centella is dried sterilization
Enter acidic alcohol solvent, the ultrasound 55min at 62 DEG C obtains the Gotu Kola P.E after filtering, drying；
The method of the culture enrichment procedure of islet cells as follows is prepared: will extract obtained islet cells by 1
×105The density of a/ml is inoculated in the culture dish equipped with the islet cell culture base, cultivates 7 days at 37 DEG C of constant temperature, so
The continuation of replacement islet cell culture base is cultivated at 37 DEG C of constant temperature afterwards, culture to the 5th generation, the islet cells after being proliferated, and
Record the culture proliferative conditions of cell.
Comparative example 1
Comparative example 1 difference from example 1 is that, islet cell culture base be RPMI-1640 culture medium, do not add remaining
Ingredient；Remaining method and step are same as Example 1.
Comparative example 2
Comparative example 2 difference from example 1 is that, replaced in islet cell culture base with the apple extract of conventional commercial
In generation, remaining method and step were same as Example 1 by apple extract made from 1 the method for embodiment.
Comparative example 3
Comparative example 3 difference from example 1 is that, do not added in islet cell culture base by 1 the method system of embodiment
The apple extract obtained, remaining method and step are same as Example 1.
Comparative example 4
Comparative example 4 difference from example 1 is that, in islet cell culture base with the Gotu Kola P.E of conventional commercial come
For substitution by Gotu Kola P.E made from 1 the method for embodiment, remaining method and step are same as Example 1.
Comparative example 5
Comparative example 5 difference from example 1 is that, do not added in islet cell culture base by 1 the method system of embodiment
The Gotu Kola P.E obtained, remaining method and step are same as Example 1.
The quantitative comparison of embodiment 1 and 1 ~ 5 islet B cell of comparative example, the results are shown in Table 1.
The islet B cell quantitative comparison of 1 embodiment of table and comparative example
As seen from the data in Table 1, embodiment 1 is best-of-breed technology scheme, and it is thin that obtained pancreas islet B is extracted and be proliferated through 1 scheme of embodiment
Born of the same parents' number is most；From embodiment 1 and comparative example 1 as it can be seen that if being only islet cell culture base, gained with RPMI-1640 culture medium
Islet B cell number after proliferation is far fewer than embodiment 1；By the comparison of embodiment 1 and comparative example 2 ~ 3 as it can be seen that if islet cells
It substitutes by apple extract made from 1 the method for embodiment or does not add with the apple extract of conventional commercial in culture medium
Add by apple extract made from 1 the method for embodiment, the islet B cell number after gained proliferation is also less than embodiment 1；From
Embodiment 1 can be seen that with comparative example 4 ~ 5, be substituted with the Gotu Kola P.E of conventional commercial by implementation in islet cell culture base
It is not added in Gotu Kola P.E made from 1 the method for example or islet cell culture base by made from 1 the method for embodiment
Gotu Kola P.E, for technical effect also without embodiment 1, finally obtained cell proliferating number can not show a candle to embodiment more than 1.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention
Range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Claims (10)
1. extraction and the enrichment procedure of a kind of islet cells, which is characterized in that extraction step and pancreas islet comprising islet cells are thin
The culture enrichment procedure of born of the same parents.
2. the extraction of islet cells and enrichment procedure according to claim 1, which is characterized in that the extraction of the islet cells
Realize that the cleaning solution includes: Hank ' s liquid, apple extract, glycyrrhiza glabra root mention by a kind of cell tissue cleaning solution
Take object；The content of the apple extract is the 5 ~ 9% of Hank ' s liquid quality；The content of the glycyrrhiza glabra root extract is
The 3 ~ 6% of Hank ' s liquid quality.
3. the extraction of islet cells and enrichment procedure according to claim 1, which is characterized in that the culture of the islet cells
Proliferation realizes that the islet cell culture base includes: RPMI-1640 culture medium, DMEM/ by a kind of islet cell culture base
F12 culture medium, fetal calf serum, the 3- hydroxyl xylose glucose of phloretin -2 ' glycosides, mycillin solution (penicillin 10000U/ml,
Streptomysin 10000ug/ml), apple extract, Gotu Kola P.E.
4. the extraction of islet cells and enrichment procedure according to claim 1, which is characterized in that the DMEM/F12 culture medium
Content be RPMI-1640 culture medium quality 30 ~ 45%；The content of fetal calf serum be RPMI-1640 culture medium quality 6 ~
10%；The content of the 3- hydroxyl xylose glucose glycosides of phloretin -2 ' is the 0.5 ~ 1% of RPMI-1640 culture medium quality；Mycillin is molten
The content of liquid (penicillin 10000U/ml, streptomysin 10000ug/ml) is the 0.4 ~ 0.9% of RPMI-1640 culture medium quality；Apple
The content of berry extract is the 2 ~ 4.5% of RPMI-1640 culture medium quality；The content of Gotu Kola P.E is RPMI-1640 culture
The 1 ~ 2.5% of matrix amount.
5. the extraction of any islet cells and enrichment procedure according to claim 1 ~ 4, which is characterized in that the apple
Extract is prepared by the inclusion of the method for following steps: smashing to pieces after apple is cleaned, volume is added with solid-liquid ratio 1:1.5 ~ 2
The ethanol water that score is 60 ~ 80% seals 50 ~ 80min of ultrasonic treatment with 350 ~ 450W of power, rotates filtrate after suction filtration
Evaporation, then the apple extract is obtained after macroreticular resin separates.
6. extraction and the enrichment procedure of islet cells according to claim 2, which is characterized in that the Gotu Kola P.E
It is prepared by the inclusion of the method for following steps: being ground after centella is dried sterilization, acidic alcohol solvent is added,
50 ~ 60min of ultrasound at 60 ~ 65 DEG C obtains the Gotu Kola P.E after filtering, drying.
7. extraction and the enrichment procedure of islet cells according to claim 2, which is characterized in that the glycyrrhiza glabra root
Extract is prepared by the inclusion of the method for following steps: grinding after glycyrrhiza glabra root is dried sterilization, hydrochloric acid is added
Alcohol solvent, 80 ~ 90min of ultrasound at 70 ~ 75 DEG C obtain the glycyrrhiza glabra root extract after filtering, drying.
8. extraction and the enrichment procedure of islet cells according to claim 1, which is characterized in that the extraction of islet cells is logical
It crosses the method comprised the following steps to realize: choosing the healthy mice pancreas of the just non-lactation of birth, remove coating, blood vessel and connective group
After knitting, it is placed in 5 ~ 7h in the cell tissue cleaning solution at 3 ~ 6 DEG C, is then that pancreas islet is separated with its hetero-organization by mechanical lapping,
Islet cells is obtained after glucan is interrupted density gradient centrifugation again.
9. extraction and the enrichment procedure of islet cells according to claim 1, which is characterized in that the culture of islet cells increases
It grows method to be prepared by the inclusion of the method for following steps: obtained islet cells will be extracted by 1 × 105The density of a/ml
It is inoculated in the culture dish equipped with the islet cell culture base, is cultivated 4 ~ 10 days at 35 ~ 38 DEG C of constant temperature, then replace pancreas islet
Cell culture medium continuation is cultivated at 35 ~ 38 DEG C of constant temperature, the islet cells after being proliferated.
10. extraction and the enrichment procedure of islet cells according to claim 1, which is characterized in that the islet cells
For islet B cell.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110063968A (en) * | 2019-04-18 | 2019-07-30 | 朗姿赛尔生物科技（广州）有限公司 | A kind of method that specific stem cells repair lesion pancreas islet |
-
2019
- 2019-04-24 CN CN201910334359.5A patent/CN110172439A/en not_active Withdrawn
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110063968A (en) * | 2019-04-18 | 2019-07-30 | 朗姿赛尔生物科技（广州）有限公司 | A kind of method that specific stem cells repair lesion pancreas islet |
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Address after: Building F2, 39 Ruihe Road, Huangpu District, Guangzhou City, Guangdong Province 510000 Applicant after: Lancy Purcell Biotechnology (Guangzhou) Co.,Ltd. Address before: 510000 Room D09, 4th Floor, 131 Airport Road, Baiyun District, Guangzhou City, Guangdong Province Applicant before: Lancy Purcell Biotechnology (Guangzhou) Co.,Ltd. |
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Application publication date: 20190827 |