CN103255106A - Autologous hematopoietic stem cell technology used for sub-health treatment - Google Patents
Autologous hematopoietic stem cell technology used for sub-health treatment Download PDFInfo
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- CN103255106A CN103255106A CN2012100353004A CN201210035300A CN103255106A CN 103255106 A CN103255106 A CN 103255106A CN 2012100353004 A CN2012100353004 A CN 2012100353004A CN 201210035300 A CN201210035300 A CN 201210035300A CN 103255106 A CN103255106 A CN 103255106A
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Abstract
The invention provides a technology which separates and purifies autologous blood-borne cells of sub-health people to cultivate stem cells being capable of being used for sub-health treatment, and relates to a field of basic medicine and health care medicine. The technology comprises taking the blood-borne cells from the sub-health people to separate and purify the stem cells, performing amplification culturing in a specific culture system, and then treating the sub-health people through intravenous transfusion after a total number of the stem cells reaches a certain number. The hematopoiesis stem cell can rebuild immunization system functions of the body and thus can be used for rehabilitation of the sub-health people. The culture amplification system can rapidly amplify the stem cells and maintain characteristics of the stem cells, type-matching is not needed during transplant, and the technology is free of toxic and side effects and adverse responses for healthcare or treatment of the sub-health people and good in effect. Meanwhile, being different from other sub-health treatment methods, such as traditional Chinese medicine therapy, nature therapy, exercise, nutritional therapy, etc., the autologous hematopoietic stem cell can substitute body cells after the transplant, and provide a good rehabilitation approach for the sub-health people.
Description
Technical field
The present invention utilizes from the cultivation of body hemocyte to amplify the stem cell that can be used for the subhealth state treatment, relate to preclinical medicine, health science field.
Background technology
" subhealth state " refers to the low state of a kind of physiological function between health and disease, mainly shows as the uncomfortable phenomenon of obvious body and mind to occur but body non-structure and metabolism unusual.Sub-health state does not have obvious clinical symptom, can not make a definite diagnosis with the relevant clinical diagnosis index.The performance of common " subhealth state " state: weak all over, tired easily, thought laxes, headache, tinnitus, eyestrain, it is dizzy to have a stuffy nose, foreign body sensation in the throat, hands and feet coolness numbness, constipation, neck shoulder stiffness, the vexed discomfort of stomach, dyssomnia, shortness of breath and palpitation, carsick easily, play dark and dim eyesight immediately, the unplessantness displeasure etc. of having got up morning.
At present, subhealth state by medical circle be known as be with acquired immune deficiency syndrome (AIDS) arranged side by side 21 century human health the formidable enemy, the predictability investigation that the World Health Organization announces recently shows that the whole world is because sub-health population overall proportion psychological, that physical efficiency stress wait factor to cause has accounted for 75%; In China, the sub-health state crowd accounts for 70% of China's population.About the subhealth state etiology, current popular be immunological theory and free radical theory.The method that the treatment of subhealth state is used always at present has tcm therapy, naturopathy, kinesitherapy, trophotherapy etc., and these therapies have certain effect, but can not improve the sub-health population physical appearance well.
(Stem Cell is that a class has self-replacation, highly increment and the cell colony of multidirectional differentiation potential SC) to stem cell.They can keep the size of self cell mass by cell fission, and can further break up again simultaneously becomes various histocyte, thereby constitute the histoorgan of the various complexity of body.According to development time, stem cell can be divided into embryonic stem cell and adult stem cell, differentiation potential according to stem cell can be divided into hematopoiesis and non-hematopoietic stem cell again, and non-hematopoietic stem cell comprises fat stem cell, neural stem cell, skin is dried, liver is done, heart is done, reproduction is done, muscle is done etc.; Hemopoietic stem cell comprises that marrow stem, periphery are done, cord blood stem cell, tire liver blood stem cell.Since the seventies in 20th century, bone marrow transplantation and autologous peripheral blood stemcell transplant are being obtained good and clinical curative effect aspect hemopathy, solid tumor, the heredopathia.1988, Frenchman Gluckman successfully carried out the first umbilical cord blood hematopoietic stem cell transplanting in the world to 5 years old Fanconi anemia infant, and after this, method, U.S., Australia etc. have completed successfully this type of stem cell transplantation of several examples in succession.The USA New York Blood Center is transplanted in the umbilical cord blood hematopoietic stem cell of the same intercellular that 1993 that finish non-blood relationship that HLA is harmonious respectively and HLA are not harmonious, and all succeeds.Stem cell not only manifests martial prowess in the treatment of disease in the blood system, in nervous system disorders, bone and joint diseases, cardiovascular disorder, metabolic disease such as diabetes, autoimmune disorder, and at aspects such as subhealth state treatment, anti-ageing, beauty treatments good application is arranged.Studies show that both at home and abroad peripheral blood contains the hemopoietic stem cell than horn of plenty, the multiplication capacity of these stem cells is stronger, and can be divided into hematopoietic cell, immunocyte etc. and can generate correlation factor.In the leukemia transplantation treatment, peripheral blood can promote the reconstruction of immunologic function, adopts simultaneously not only can remove from transplanting from the body hemocytoblast and joins type, can also remove the detection of virus from.Therefore, provide new way from the body hemocytoblast for the rehabilitation of sub-health population.
No matter yet be for treatment of diseases, how the or rehabilitation of subhealth state obtains sufficient hemopoietic stem cell and is the difficult problem that medical profession for a long time faces.Early stage amplifying candidate stem cell adopts various combination mode amplifying candidate stem cell in vitro such as combined utilization hemopoieticgrowth factor such as SCF, FL, TPO, GM-CSF, G-CSF, IL-3, IL-6, Lif, bFGF more, cells expanded is inadequate, amplification back cell may have problems such as directed differentiation ability but all exist, so its clinical application is limited to.The present invention adopts mescenchymal stem cell as the place mat cell, is equipped with nutritional factor simultaneously and cultivates amplifying candidate stem cell, for subhealth state person's transplantation treatment provides good technical.
Summary of the invention
The purpose of this invention is to provide a kind of subhealth state treatment and use the hemopoietic stem cell technology, it is to obtain the sub-health population peripheral blood, and is the place mat cell with the umbilical cord mesenchymal stem cells in 100 grades of GMP factory buildings, adds relevant nutritional factor cultivation amplifying candidate stem cell.After quality inspection is qualified, feed back again and give subhealth state person, reach the effect of its rehabilitation.This kind stem cell transplantation need not to join type, need not to detect specific virus, and is simple, safe and reliable, for subhealth state person's rehabilitation provides good method.
Embodiment
Specifying subhealth state treatment autologous stem cell technology of the present invention by present embodiment, is that example explanation order of concrete step is as follows with the autologous peripheral blood stem cell here:
(1) stroma cell is cultivated, sign puerpera's umbilical cord and contribute letter of consent, under the delivery room aseptic condition, obtain puerpera's umbilical cord, in 100 grades of laminar flow hood in 36 hours inherent G M P workshops the blood vessel of umbilical cord tissue is removed, be cut into the tissue block of 1mm3 size, be inoculated in the T75 Tissue Culture Flask, the DMEM complete culture solution also adds nutritional factor, i.e. 10ng/ml ITS (Regular Insulin, Transferrins,iron complexes, selenium), 100u/ml penicillin, the 100u/ml Streptomycin sulphate, 20ng/ml of rEGF), 20ng/ml of rPDGF-bb), 10ng/ml of LIF, 20ng/ml of bFGF is positioned over 37 ℃, cultivate in the incubator of 5%CO2.After 7-10 days, mescenchymal stem cell grows from the tissue block edge, changes liquid, and the extra-nutrition factor.Cell degree of converging reaches at 80% o'clock, and stem cell is also filtered with 0.25% trysinization, the cell suspension centrifugal 6min of 800r/min, resuspended with the DMEM complete culture solution, and go down to posterity in T 175ml culturing bottle (P1 generation), the extra-nutrition factor, going down to posterity afterwards passes according to 1: 3 ratio.
(2) autologous stem cell is cultivated, extract 10 milliliters of subhealth state person's peripheric venous bloods under the sterile state, with heparin (20U/mL) anti-freezing, in 100 grades of GMP factory buildings, the application of stem cells separating machine adopts the Ficoll density gradient centrifugation to separate stem cell (CD34+ cell) in the peripheral blood.Taking heparin anti-freezing venous blood and equivalent Hank ' s liquid or the abundant mixing of DMEM perfect medium add 1.077g/mL Ficoll lymphocyte separation medium 3mL, note keeping clearly interface, the centrifugal 2000rpm of level * 20 minutes.Be divided into three layers in the pipe of centrifugal back, the upper strata is blood plasma and Hank ' s liquid, and lower floor is mainly red corpuscle and granulocyte.The middle level is lymphocyte separation medium, and the narrow band of white cloud and mist layer based on mononuclearcell arranged at the interface in last, middle level, and mononuclearcell comprises lymphocyte and monocyte, in addition, also contains thrombocyte.Be inserted into the cloud and mist layer with capillary vessel, draw mononuclearcell, insert in another short tube, add 5 times with Hank ' s liquid or the DMEM perfect medium of upper volume, 1500rpm * 10 minute, washed cell three times, and be resuspended in the DMEM perfect medium.Monocyte is with 1 * 10
5Cells/mL density is planted in T175 culturing bottle (wherein having contained P2 for the matrix umbilical cord mesenchymal stem cells that has been in 60% cell degree of converging), the extra-nutrition factor: 50ng/mL of SCF, 10U/Ml of TPO, 50ng/mL of G-CSF, 2.7% methylcellulose gum, 100ng/mLGM-CSF, 1 * 500UI/ml of IL-1,10ng/ml of IL-3,10ng/ml of Flt3-L, changed liquid once in per three days.
(3) cultivation of going down to posterity of hemopoietic stem cell is taken out the mescenchymal stem cell culture supernatant earlier, the centrifugal 6min of 800r/min, and centrifuged supernatant refilters out decon, the resuspended stem cell precipitation of a little filtrate.Another P 2 generation umbilical cord mesenchymal stem cells that is in 60% cell degree of converging is cultivated (T 175ml culturing bottle) supernatant liquid filtering, 1/3rd add former culturing bottle, former hemopoietic stem cell nutrient solution 1/3rd is used for resuspended hemopoietic stem cell, replenish in addition 1/3rd fresh mediums and the factor that supplements the nutrients (fill do and hematopoiesis do cultivate nutritional factor), be positioned over 37 ℃, the cultivation of 5%CO2 incubator saturated humidity, every 3d changes once.
(4) autologous stem cell is transplanted, and the hemopoietic stem cell that increases is carried out quality inspection: the stem cell centrifuged supernatant is bacterium smear staining, microorganism culturing (anerobe, aerophil cultivation, fungi and mould), karyocyte counting, stem cell vigor counting, CD34
+Cell counting, stem cell colonies form experiment.Sign insider's letter of consent before the stem cell transplantation, the autologous peripheral blood stem cell is after quality inspection is qualified, with 3X10
5The stem cell dose of/KG feeds back to subhealth state person.Remaining stem cell cryopreserving (autologous stem cells for preparing is added 10%DMSO and 1%BSA, deposit-196 ° of liquid nitrogen containers in and preserve).
Claims (6)
1. the autologous stem cell technology is used in the subhealth state treatment, it is characterized in that it is undertaken by following step order:
(1) stroma cell is cultivated, and obtains puerpera's umbilical cord under the aseptic condition, handles umbilical cord in the GMP workshop and cultivates umbilical cord mesenchymal stem cells.
(2) autologous stem cell is cultivated, and obtains peripheric venous blood under aseptic condition, adopts the Ficoll density gradient centrifugation to separate peripheral hematopoietic stem cells, and to be inoculated in the umbilical cord mesenchymal stem cells be the culture system of matrix.
(3) cultivation of going down to posterity of hemopoietic stem cell is taken out the mescenchymal stem cell culture supernatant earlier, and is centrifugal, and attached cell is inoculated in another umbilical cord mesenchymal stem cells and cultivates.
(4) autologous stem cell is transplanted, and the hemopoietic stem cell that increases is passed through vein transplantation subhealth state person.
2. the autologous stem cell technology of using is treated in subhealth state according to claim 1, it is characterized in that: described step order (1) stroma cell is cultivated, sign umbilical cord with the puerpera and contribute letter of consent and obtain puerpera's umbilical cord under aseptic condition, the fragment cultivation is removed and be cut in 100 grades of GMP workshops with blood vessel and the adventitia of umbilical cord: DMEM complete culture solution, 10ng/ml ITS (Regular Insulin, Transferrins,iron complexes, selenium), 100u/ml penicillin, 100u/ml Streptomycin sulphate, 20ng/ml of rEGF, 20ng/ml of rPDGF-bb, 10ng/ml of LIF, 20ng/ml of bFGF.Stem cell degree of converging reaches at 80% o'clock and goes down to posterity.
3. the autologous stem cell technology of using is treated in subhealth state according to claim 1, it is characterized in that: described step order (2) autologous stem cell is cultivated, extract 10 milliliters in subhealth state person's venous blood under the aseptic condition, heparin (20U/mL) anti-freezing is in 100 grades of GMP factory buildings Ficoll density gradient centrifugation separate stem cells (CD34+ cell).Be inserted into the cloud and mist layer with capillary vessel, draw mononuclearcell washing three times, with 1 * 10
5Cells/mL density is planted in T175 culturing bottle (wherein containing P2 matrix umbilical cord mesenchymal stem cells), the extra-nutrition factor: 50ng/mL of SCF, 10U/Ml of TPO, 50ng/mL of G-CSF, 2.7% methylcellulose gum, 100ng/mL GM-CSF, 1 * 500UI/ml of IL-1,10ng/ml of IL-3,10ng/ml of Flt3-L, changed liquid once in per three days.
4. the autologous stem cell technology of using is treated in subhealth state according to claim 1, it is characterized in that: the cultivation of going down to posterity of described step order (3) hemopoietic stem cell, earlier the hemopoietic stem cell nutrient solution is taken out, centrifugal, filter, another P2 that is in 60% cell degree of converging is filtered for the umbilical cord mesenchymal stem cells culture supernatant.Each 1/3rd adds above-mentioned P2 and fills driedly between generation to fill dried nutrient solution filtered liquid and fresh medium between former hemopoietic stem cell nutrient solution filtered liquid, another P2, and 1/3rd of hemopoietic stem cell is used for going down to posterity and adding relevant nutritional factor.
5. the autologous stem cell technology of using is treated in subhealth state according to claim 1, it is characterized in that: described step order (4) autologous stem cell is transplanted, and the autologous stem cell of amplification carries out quality inspection and comprises that microorganism checking, karyocyte counting, stem cell vigor counting, CD34+ cell counting, stem cell colonies form experiment.The qualified stem cell of quality inspection is in subhealth state person's hand back vein infusion, and Transplanted cells dosage is 3X10
5/ KG.Remaining stem cell cryopreserving deposits-196 ° of liquid nitrogen containers in 10%DMSO and 1%BSA and preserves.
6. the autologous stem cell technology is used in subhealth state treatment according to claim 1, and wherein said autologous stem cell comprises marrow hemopoietic stem cells, peripheral blood hematopoietic stem cells, cord blood stem cell, all is fit to the technology of the present invention scope.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103385890A (en) * | 2013-07-17 | 2013-11-13 | 崔澂 | A preparation method for an anti-aging biological cell preparation |
| CN108888636A (en) * | 2018-08-14 | 2018-11-27 | 东营凤起生物科技发展有限公司 | A method for the treatment of diabetes and atherosclerosis |
-
2012
- 2012-02-16 CN CN2012100353004A patent/CN103255106A/en active Pending
Non-Patent Citations (5)
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| TIKI BAKHSHI,ET AL: "Mesenchymal stem cells from theWharton’s jelly of umbilical cord segments provide stromal support for the maintenance of cord blood hematopoietic stem cells during long-term ex vivo culture", 《TRANSFUSION》 * |
| YI ZHANG，ET AL: "Human placenta-derived mesenchymal progenitor cells support culture expansion of long-term culture-initiating cells from cord blood CD34＋ cells", 《EXPERIMENTAL HEMATOLOGY》 * |
| YUN KYUNG JANG，ET AL: "Mesenchymal stem cells feeder layer from human umbilical cord blood for ex vivo expanded growth and proliferation of hematopoietic progenitor cells", 《ANN HEMATOL》 * |
| 徐燕 等: "人脐带间充质干细胞分离培养条件的优化及其生物学特性", 《中国组织工程研究与临床康复》 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103385890A (en) * | 2013-07-17 | 2013-11-13 | 崔澂 | A preparation method for an anti-aging biological cell preparation |
| CN103385890B (en) * | 2013-07-17 | 2015-09-23 | 崔澂 | A kind of preparation method of anti-ageing biological cell preparation |
| CN108888636A (en) * | 2018-08-14 | 2018-11-27 | 东营凤起生物科技发展有限公司 | A method for the treatment of diabetes and atherosclerosis |
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Application publication date: 20130821 |