CN114891746A - Preparation method of canine whole blood hematopoietic stem cells - Google Patents
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- 210000004369 blood Anatomy 0.000 title claims abstract description 47
- 239000008280 blood Substances 0.000 title claims abstract description 47
- 210000003958 hematopoietic stem cell Anatomy 0.000 title claims abstract description 45
- 241000282465 Canis Species 0.000 title claims abstract description 14
- 238000002360 preparation method Methods 0.000 title claims abstract description 8
- 241000282472 Canis lupus familiaris Species 0.000 claims abstract description 79
- 238000000034 method Methods 0.000 claims abstract description 19
- 238000001514 detection method Methods 0.000 claims abstract description 15
- 210000002966 serum Anatomy 0.000 claims abstract description 9
- 238000002965 ELISA Methods 0.000 claims abstract description 8
- 229960005486 vaccine Drugs 0.000 claims abstract description 8
- 239000003795 chemical substances by application Substances 0.000 claims description 25
- 230000001483 mobilizing effect Effects 0.000 claims description 25
- 238000002347 injection Methods 0.000 claims description 12
- 239000007924 injection Substances 0.000 claims description 12
- 230000003203 everyday effect Effects 0.000 claims description 10
- 229940109850 royal jelly Drugs 0.000 claims description 4
- 101000746367 Homo sapiens Granulocyte colony-stimulating factor Proteins 0.000 claims description 2
- 244000052769 pathogen Species 0.000 abstract description 4
- 238000003745 diagnosis Methods 0.000 abstract description 3
- 239000010836 blood and blood product Substances 0.000 abstract description 2
- 229940125691 blood product Drugs 0.000 abstract description 2
- 230000001717 pathogenic effect Effects 0.000 abstract 1
- 210000003743 erythrocyte Anatomy 0.000 description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 5
- 238000002649 immunization Methods 0.000 description 5
- 230000003053 immunization Effects 0.000 description 5
- 102100036475 Alanine aminotransferase 1 Human genes 0.000 description 3
- 108010082126 Alanine transaminase Proteins 0.000 description 3
- 102000009027 Albumins Human genes 0.000 description 3
- 108010088751 Albumins Proteins 0.000 description 3
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 3
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 3
- 108010003415 Aspartate Aminotransferases Proteins 0.000 description 3
- 102000004625 Aspartate Aminotransferases Human genes 0.000 description 3
- 102000001554 Hemoglobins Human genes 0.000 description 3
- 108010054147 Hemoglobins Proteins 0.000 description 3
- 238000008050 Total Bilirubin Reagent Methods 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 238000005534 hematocrit Methods 0.000 description 3
- 210000000265 leukocyte Anatomy 0.000 description 3
- 238000004820 blood count Methods 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 238000011134 hematopoietic stem cell transplantation Methods 0.000 description 2
- 210000005259 peripheral blood Anatomy 0.000 description 2
- 239000011886 peripheral blood Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 208000025174 PANDAS Diseases 0.000 description 1
- 208000021155 Paediatric autoimmune neuropsychiatric disorders associated with streptococcal infection Diseases 0.000 description 1
- 240000004718 Panda Species 0.000 description 1
- 235000016496 Panda oleosa Nutrition 0.000 description 1
- PNNCWTXUWKENPE-UHFFFAOYSA-N [N].NC(N)=O Chemical compound [N].NC(N)=O PNNCWTXUWKENPE-UHFFFAOYSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000010241 blood sampling Methods 0.000 description 1
- 238000011072 cell harvest Methods 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 230000002354 daily effect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 208000014951 hematologic disease Diseases 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/02—Breeding vertebrates
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Abstract
The invention discloses a preparation method of canine whole blood hematopoietic stem cells, belonging to the technical field of blood products. A preparation method of canine whole blood hematopoietic stem cells comprises the following steps: the method comprises the following steps: matching SPF dogs, wherein the blood type of the SPF dogs is negative blood, the SPF dogs need to be immunized by a conventional quintuplet vaccine and are detected by adopting an indirect ELISA method, and the antibody titer of the SPF dogs reaches 2 9 Above, the SPF dog needs to be matched with a sick dog; step two: carrying out physical examination on the dogs, and periodically collecting venous blood for hematology and serum biochemical detection before blood collection of the matched dogs; the experimental dog is an SPF dog without special pathogen, and has great advantages in the aspect of safety performance, so the prepared blood has good safety performance, hematopoietic stem cells are provided for other sick dogs by taking the SPF greyhound as a volunteer, the safety of blood transfusion is improved, the cure rate of the sick dogs is greatly improved, and the blood transfusion system plays a role in the critical point of diagnosis and treatment of the sick dogsThe intended function.
Description
Technical Field
The invention relates to the field of blood products, in particular to a preparation method of canine whole blood hematopoietic stem cells.
Background
Hematopoietic stem cell transplantation, which has been developed for about half a century, has been widely used for the treatment of hematological diseases, and more than 10 ten thousand patients have been subjected to various hematopoietic stem cell transplants worldwide. The treatment of malignant diseases by hematopoietic stem cell transplantation accounts for most of clinical cases, and the invention adopts a method for obtaining hematopoietic stem cells from peripheral blood of SPF dogs to provide perfect hematopoietic stem cells for sick dogs.
Disclosure of Invention
The present invention is directed to a method for producing canine whole blood hematopoietic stem cells, which solves the problems of the background art described above.
In order to achieve the purpose, the invention adopts the following technical scheme:
a preparation method of canine whole blood hematopoietic stem cells comprises the following steps:
the method comprises the following steps: the canine matching is carried out, the canine blood type is negative blood, the canine needs to be immunized by a conventional quintuplet vaccine, an indirect ELISA method is adopted for detection, and the antibody titer reaches 2 9 Above, the dog needs to be matched with a sick dog;
step two: carrying out physical examination on the dogs, and periodically collecting venous blood for hematology and serum biochemical detection before blood collection of the matched dogs, wherein each index is in a normal range;
step three: the dog hematopoietic stem cell collection is characterized in that an mobilizing agent is injected every day before the hematopoietic stem cells are collected, and the dog is fed with royal jelly before the hematopoietic stem cells are collected, so that the content of the hematopoietic stem cells is increased.
Preferably, step one, step two and step three are all performed in a completely sterile closed chamber.
Preferably, the dog is an SPF dog, including greyhound, beagle dog, dobby dog, boxer, german shepherd dog, etc., the SPF dog is between 2-4 years of age and between 20-40 kg in weight.
Preferably, the mobilizing agent is a recombinant human granulocyte colony stimulating factor injection: rhG-CSF.
Preferably, the mobilizing agent is injected in an amount of 150 μ g/kg/day subcutaneously 1 time per day for five consecutive days.
Preferably, the fifth day of injection of the mobilizer is the first hematopoietic stem cell harvest.
Preferably, the second collection is performed if the first collection is insufficient for hematopoietic stem cells.
Compared with the prior art, the invention provides a preparation method of canine whole blood hematopoietic stem cells, which has the following beneficial effects:
the experimental dog is an SPF dog without special pathogens, and has great advantages in the aspect of safety performance, so that the prepared blood has good safety performance, and the SPF greyhound dog is used as a volunteer to provide hematopoietic stem cells for other sick dogs, so that the blood transfusion safety is improved, the cure rate of the sick dogs is greatly improved, and the blood transfusion system plays an important role in diagnosis and treatment of the sick dogs.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments.
Example 1:
selecting negative Ti-Baker of 2-4 years old with weight of 20-40 kg;
the conventional quintuplet vaccine is used for immunization, an indirect ELISA method is used for detection, and the antibody titer reaches over 29;
the SPF dog needs to be matched with a sick dog;
before blood collection of a matched dog, venous blood needs to be collected regularly for hematology and serum biochemical detection, and all indexes are in a normal range;
peripheral blood was taken directly.
Example 2:
selecting negative Ti-Baker of 2-4 years old with weight of 20-40 kg;
the conventional quintuplet vaccine is used for immunization, an indirect ELISA method is used for detection, and the antibody titer reaches over 29;
the panda needs to be matched with a sick dog;
before blood collection of a matched dog, venous blood needs to be collected regularly for hematology and serum biochemical detection, and all indexes are in a normal range;
injecting mobilizing agent (rhG-CSF) every day before collecting hematopoietic stem cells;
the injection amount of the mobilizing agent is 150 mug/kg/day, and the mobilizing agent is subcutaneously injected for 1 time every day for five consecutive days;
collecting hematopoietic stem cells for the first time on the fifth day, and if the hematopoietic stem cells collected for the first time are insufficient, collecting for the second time.
Example 3:
selecting an SPF negative greyhound dog of 2-4 years old, wherein the weight is 20-40 kg;
the conventional quintuplet vaccine is used for immunization, an indirect ELISA method is used for detection, and the antibody titer reaches over 29;
the SPF smart dog needs to be matched with a sick dog;
before blood collection of a matched dog, venous blood needs to be collected regularly for hematology and serum biochemical detection, and all indexes are in a normal range;
injecting mobilizing agent (rhG-CSF) every day before collecting hematopoietic stem cells;
the injection amount of the mobilizing agent is 150 mug/kg/day, and the mobilizing agent is subcutaneously injected for 1 time every day for five consecutive days;
collecting hematopoietic stem cells for the first time on the fifth day, and if the hematopoietic stem cells collected for the first time are insufficient, collecting for the second time.
Example 4:
selecting an SPF negative greyhound dog of 2-4 years old, wherein the weight is 20-40 kg;
the conventional quintuplet vaccine is used for immunization, an indirect ELISA method is used for detection, and the antibody titer reaches over 29;
the SPF smart dog needs to be matched with a sick dog;
before blood collection of a matched dog, venous blood needs to be collected regularly for hematology and serum biochemical detection, and all indexes are in a normal range;
injecting mobilizing agent (rhG-CSF) every day before collecting hematopoietic stem cells;
the injection amount of the mobilizing agent is 300 mug/kg/day, and the mobilizing agent is subcutaneously injected for 1 time every day for five consecutive days;
collecting hematopoietic stem cells for the first time on the fifth day, and if the hematopoietic stem cells collected for the first time are insufficient, collecting for the second time.
Example 5:
selecting an SPF negative greyhound dog of 2-4 years old, wherein the weight is 20-40 kg;
the conventional quintuplet vaccine is used for immunization, an indirect ELISA method is used for detection, and the antibody titer reaches over 29;
the SPF smart dog needs to be matched with a sick dog;
before blood collection of a matched dog, venous blood needs to be collected regularly for hematology and serum biochemical detection, and all indexes are in a normal range;
injecting mobilizing agent (rhG-CSF) every day before collecting hematopoietic stem cells;
the injection amount of the mobilizing agent is 150 mug/kg/day, and the mobilizing agent is subcutaneously injected for 1 time every day for five consecutive days;
collecting hematopoietic stem cells for the first time on the fifth day, and performing the second collection if the hematopoietic stem cells collected for the first time are insufficient;
the dogs were fed royal jelly daily before hematopoietic stem cells were harvested.
Data processing and analysis
Before injection of mobilizing agent, 20 Beagle dogs were subjected to venous blood sampling in a fasting state, and total white blood cell count (WBC), Hemoglobin (HGB), total red blood cell count (RBC), Hematocrit (HCT), mean red blood cell volume (MCV), mean red blood cell Hb (MCH), mean red blood cell Hb concentration (MCHC), and Platelets (PLT) were measured using a hemocytometer. Separating serum, and measuring biochemical indexes such as blood Glucose (GLU), urea nitrogen (BUN), total bilirubin (T-BIL), Total Cholesterol (TCH), Total Protein (TP), Albumin (ALB), aspartate Aminotransferase (AST), alanine Aminotransferase (ALT), alkaline phosphatase (ALP), etc. with a full-automatic biochemical analyzer.
TABLE 1 Beagle dog blood normative values prior to injection of mobilizing agent
| Index (I) | Male(s) | Female |
| WBC/109 L-1 | 11.57±2.91 | 12.72±2.46 |
| RBC/1012 L-1 | 6.64±1.94 | 7.23±1.74 |
| HGB/g L-1 | 152.84±12.36 | 167.53±27.69 |
| HCT/% | 18.14±21.65 | 17.64±21.37 |
| MCV/fL | 65.66±12.27 | 58.73±12.97 |
| MCH/pg | 22.34±1.46 | 23.57±2.62 |
| MCHC/g L-1 | 358.25±58.02 | 401.62±54.06 |
| PLT/109 L-1 | 278.46±58.30 | 263.28±60.24 |
TABLE 2 Beagle dog blood Biochemical values before injection of mobilizing agent
| Index (I) | Male sex | Female |
| GLU/mmol L-1 | 4.75±0.54 | 4.75±0.46 |
| CRE/umol L-1 | 60.78±7.95 | 93.85±7.48 |
| BUN/mmol L-1 | 1.95±0.54 | 2.45±0.86 |
| T-BIL/umol L-1 | 7.69±2.47 | 6.45±2.95 |
| TCH/mmol L-1 | 4.55±0.96 | 4.59±0.68 |
| TP/g L-1 | 50.31±4.13 | 45.31±4.86 |
| ALB/g L-1 | 27.52±2.00 | 24.20±4.23 |
| ALT/U L-1 | 28.43±10.25 | 31.25±12.25 |
| AST/U L-1 | 21.23±5.05 | 21.10±4.17 |
| ALP/U L-1 | 155.56±31.25 | 148.48±45.03 |
As can be seen from the above, the routine and biochemical values of the blood of Beagle dogs before injection of the mobilizing agent are both satisfactory and suitable for collection of hematopoietic stem cells.
In summary, the embodiments of the present invention provide a method for preparing canine whole blood hematopoietic stem cells, wherein the experimental dog is an SPF dog without specific pathogens, and the blood contains a large amount of antibodies, which has great advantages in safety and nutrition, and the content of hematopoietic stem cells is greatly increased by injecting mobilizing agents and feeding royal jelly. Therefore, the prepared hematopoietic stem cells not only have good safety performance, but also contain rich antibodies and have high content.
The experimental dog is an SPF dog without special pathogens, and has great advantages in the aspect of safety performance, so that the prepared blood has good safety performance, and the SPF greyhound dog is used as a volunteer to provide hematopoietic stem cells for other sick dogs, so that the blood transfusion safety is improved, the cure rate of the sick dogs is greatly improved, and the blood transfusion system plays an important role in diagnosis and treatment of the sick dogs.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (7)
1. A preparation method of canine whole blood hematopoietic stem cells is characterized by comprising the following steps:
the method comprises the following steps: matching the type of the dog, wherein the blood type of the SPF dog is negative blood, the dog needs to be immunized by a conventional quintuplet vaccine, an indirect ELISA method is adopted for detection, and the antibody titer reaches 2 9 Above, the dog needs to be matched with a sick dog;
step two: carrying out physical examination on the dogs, and periodically collecting venous blood for hematology and serum biochemical detection before blood collection of the matched dogs, wherein each index is in a normal range;
step three: the dog hematopoietic stem cell collection is characterized in that an mobilizing agent is injected every day before the hematopoietic stem cells are collected, and the dog is fed with royal jelly before the hematopoietic stem cells are collected, so that the content of the hematopoietic stem cells is increased.
2. The method of claim 1, wherein the steps one, two and three are performed in a completely sterile closed chamber.
3. The method of claim 1, wherein the dog is an SPF dog, the SPF dog comprises a greyhound, a beagle dog, a Dubinge dog, a boxer, a German shepherd dog, the SPF dog is between 2 and 4 years of age, and the weight of the dog is between 20 and 40 kg.
4. The method for preparing canine whole blood hematopoietic stem cells according to claim 1, wherein the mobilizing agent is rhG-CSF (recombinant human granulocyte colony-stimulating factor) injection.
5. The method of claim 1, wherein the amount of the mobilizing agent injected is 150 μ g/kg/day, and the mobilizing agent is injected subcutaneously 1 time a day for five consecutive days.
6. The method according to claim 5, wherein the first hematopoietic stem cell collection is performed on the fifth day of injection of the mobilizing agent.
7. The method according to claim 6, wherein the second collection is performed if the first collection is insufficient for hematopoietic stem cells.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1826134A (en) * | 2003-07-29 | 2006-08-30 | 冬姆佩股份公司 | Pharmaceutical combination of G-CSF and PLGF useful for blood stem cell |
| CN101716167A (en) * | 2009-12-08 | 2010-06-02 | 中国人民解放军军事医学科学院野战输血研究所 | Use of saturated amine compound in preparation of medicaments for mobilizing peripheral blood hematopoietic stem cells |
| US20120269774A1 (en) * | 2006-09-21 | 2012-10-25 | Medistem Laboratories, Inc | Allogeneic stem cell transplants in non-conditioned recipients |
| CN111778212A (en) * | 2020-07-08 | 2020-10-16 | 因诺伟(北京)生物医疗科技有限公司 | Preparation method and application of mobilized hematopoietic stem cell plasma exosome |
| CN112852734A (en) * | 2021-02-24 | 2021-05-28 | 河南省银丰生物工程技术有限公司 | Method for separating and identifying human peripheral blood hematopoietic stem cells |
-
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- 2021-12-21 CN CN202111571481.8A patent/CN114891746A/en active Pending
Patent Citations (5)
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|---|---|---|---|---|
| CN1826134A (en) * | 2003-07-29 | 2006-08-30 | 冬姆佩股份公司 | Pharmaceutical combination of G-CSF and PLGF useful for blood stem cell |
| US20120269774A1 (en) * | 2006-09-21 | 2012-10-25 | Medistem Laboratories, Inc | Allogeneic stem cell transplants in non-conditioned recipients |
| CN101716167A (en) * | 2009-12-08 | 2010-06-02 | 中国人民解放军军事医学科学院野战输血研究所 | Use of saturated amine compound in preparation of medicaments for mobilizing peripheral blood hematopoietic stem cells |
| CN111778212A (en) * | 2020-07-08 | 2020-10-16 | 因诺伟(北京)生物医疗科技有限公司 | Preparation method and application of mobilized hematopoietic stem cell plasma exosome |
| CN112852734A (en) * | 2021-02-24 | 2021-05-28 | 河南省银丰生物工程技术有限公司 | Method for separating and identifying human peripheral blood hematopoietic stem cells |
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| Title |
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| SANGHO KIM等: "Comparison of three mobilization protocols for peripheral blood stem cell apheresis with Spectra Optia continuous mononuclear cell protocol in healthy dogs", 《VET COMP ONCOL 》 * |
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