CN108396009A - A kind of method that the adjustment type macrophage of in vitro culture induction is used for myocardial preservation - Google Patents
A kind of method that the adjustment type macrophage of in vitro culture induction is used for myocardial preservation Download PDFInfo
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- CN108396009A CN108396009A CN201810200942.2A CN201810200942A CN108396009A CN 108396009 A CN108396009 A CN 108396009A CN 201810200942 A CN201810200942 A CN 201810200942A CN 108396009 A CN108396009 A CN 108396009A
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Abstract
The invention discloses the methods that a kind of adjustment type macrophage of in vitro culture induction is used for myocardial preservation, comprise the steps of：S1：Derived from bone marrow macrophage, culture induction adjustment type macrophage are extracted, all operations carry out under aseptic condition；S2：Prepare macrophage suspension：Adjustment type macrophage is resuspended using pancreatin digestion into physiological saline using preceding；S3：Cell transplantation.Adjustment type macrophage is proposed for myocardial preservation for the first time; adjustment type macrophage is as novel methods of myocardial protection; take into account improve myocardial preservation in the recent period and long-term effect; improve patients ' life quality; can further offer reference experience for the treatment of the ischemical reperfusion injury and organ fibrosis of a variety of organs; bone marrow aspiration takes autologous bone marrow; through in vitro culture and induction; turn out adjustment type macrophage; migrate to patient's cardiac muscle; because of its nearly Myocardial protective effects at a specified future date, has the function that improve heart function, reduce postoperative complications, reduce cardiac fibrosis at a specified future date.
Description
Technical field
The present invention relates to methods of myocardial protection technical field, specially a kind of adjustment type macrophage of in vitro culture induction
Method for myocardial preservation.
Background technology
Myocardial preservation measure at present is often confined to the intervention to inflammation damnification, lacks the intervention to fibrosis at a specified future date, and
One of an important factor for fibrosis is also influence patient's prognosis after myocardial damage, for a kind of tune of in vitro culture induction of the invention
Nodal pattern macrophage is used to solve above-mentioned drawback for the method for myocardial preservation, and the different referred to as M2b types of adjustment type macrophage are huge
Phagocyte.
Invention content
The purpose of the present invention is to provide the sides that a kind of adjustment type macrophage of in vitro culture induction is used for myocardial preservation
Method, to solve the problems mentioned in the above background technology.
To achieve the above object, the present invention provides the following technical solutions：A kind of adjustment type macrophage of in vitro culture induction is thin
The method that born of the same parents are used for myocardial preservation, this approach includes the following steps：
S1：Derived from bone marrow macrophage is extracted, culture induction adjustment type macrophage, all operations are under aseptic condition
It carries out, concrete operations are as follows：
One, bone marrow aspiration takes autologous patient marrow, external to be resuspended into RPMI-1640 culture mediums, the filtering of 200 mesh filter screens,
500g centrifuges 5min, abandons supernatant, and cell is resuspended to BMM culture mediums in erythrocyte cracked liquid splitting erythrocyte；
Two, cell is placed in 37 DEG C, 50mL/L CO2 incubators, per changing within 2-3 days liquid (fresh M-CSF need to be added by changing liquid every time), 7
It becomes ripe monocytes/macrophages (M0)；
Three, the M0 of induced maturation stimulates 48h as follows：Chicken egg white (OVA)-lgG immune complexs (15 μ g/ml OVA
With 150 μ g/ml anti ova-lgG, 37 DEG C of incubation 30min)+LPS (50ng/ml), i.e. induction is ripe adjustment type macrophage；
S2：Prepare macrophage suspension：Adjustment type macrophage is resuspended using pancreatin digestion into physiological saline using preceding,
Adjust a concentration of 1 × 107A/mL, total amount are 1 × 106A/kg；
S3：Cell transplantation, for openheart surgery under extracorporal circulatory system, in cardiac arrest, with myocardium protecting liquid, perfusion
To coronary artery, it to be used for the patient of acute coronary artery syndrome, when percutaneous percutaneous transluminal coronary stent implantation, is noted through conduit after stenter to implant
It is incident upon coronary artery.
Preferably, the adjustment type macrophage induced through above-mentioned condition, can be through the above method for openheart surgery and acute
The patient of coronary syndrome.
Compared with prior art, the beneficial effects of the invention are as follows：Adjustment type macrophage is proposed for myocardium guarantor for the first time
Shield, adjustment type macrophage are taken into account and improve myocardial preservation in the recent period and long-term effect, improve patient as novel methods of myocardial protection
Quality of life, can further offer reference experience for the treatment of the ischemical reperfusion injury and organ fibrosis of a variety of organs, need
It wants the patient of this treatment, bone marrow aspiration to take autologous bone marrow, through in vitro culture and induction, turns out adjustment type macrophage, transplant
To patient's cardiac muscle, because of its nearly Myocardial protective effects at a specified future date, reaching improves heart function, reduces postoperative complications, reduces the heart at a specified future date
The effect of dirty fibrosis.
Description of the drawings
Fig. 1 is flowage structure schematic diagram when present invention adjustment type macrophage is tested for human body myocardial preservation；
Fig. 2 is flowage structure schematic diagram when present invention adjustment type macrophage is used for animal cardiac muscle Protection；
Fig. 3 is that the present invention is used to mitigate when zoopery myocardial damage (reducing Troponin I) schematic diagram；
Fig. 4 is that the present invention is used to mitigate when zoopery myocardial damage (reducing infarct size) schematic diagram；
Fig. 5 is that the present invention is used to mitigate when zoopery myocardial damage (reducing cardiac muscle cell apoptosis) schematic diagram；
Fig. 6 is that the present invention is used to mitigate the cardiac fibrosis structural representation after myocardial ischemia-reperfusion injury when zoopery
Figure；
Fig. 7 is that the cardiac systolic function structure after improving myocardial ischemia-reperfusion injury when the present invention is used for zoopery is shown
It is intended to.
In figure：Fig. 3, which is cell therapy, can reduce the serum cTnI (Troponin I after myocardial ischemia-reperfusion injury
For myocardial injury markers), Fig. 4 is cell therapy can reduce the infarct size (darker regions after myocardial ischemia-reperfusion injury：
Non- ischemic area；Light areas：The non-infarcted region of ischemic；Light light areas：Infarcted region), Fig. 5 be cell therapy can reduce the heart
Cardiac muscle cell apoptosis (darker regions after myocardial ischemia reperfusion injury：All nucleus；Light areas：Apoptotic cell), Fig. 6 be
Cell therapy can mitigate the cardiac fibrosis (darker regions after myocardial ischemia-reperfusion injury：Collagenous fibres in heart), Fig. 7
For for M type cardiac ultrasonics.
Specific implementation mode
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation describes, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
- 7 are please referred to Fig.1, the present invention provides a kind of technical solution：A kind of adjustment type macrophage use of in vitro culture induction
In the method for myocardial preservation, comprise the steps of：
S1：Derived from bone marrow macrophage is extracted, culture induction adjustment type macrophage, all operations are under aseptic condition
It carries out, concrete operations are as follows：
One, bone marrow aspiration takes autologous patient marrow, external to be resuspended into RPMI-1640 culture mediums, the filtering of 200 mesh filter screens,
500g centrifuges 5min, abandons supernatant, and cell is resuspended to BMM culture mediums in erythrocyte cracked liquid splitting erythrocyte；
Two, cell is placed in 37 DEG C, 50mL/L CO2 incubators, per changing within 2-3 days liquid (fresh M-CSF need to be added by changing liquid every time), 7
It becomes ripe monocytes/macrophages (M0)；
Three, the M0 of induced maturation stimulates 48h as follows：Chicken egg white (OVA)-lgG immune complexs (15 μ g/ml OVA
With 150 μ g/ml anti ova-lgG, 37 DEG C of incubation 30min)+LPS (50ng/ml), i.e. induction is ripe adjustment type macrophage；
S2：Prepare macrophage suspension：Adjustment type macrophage is resuspended using pancreatin digestion into physiological saline using preceding,
Adjust a concentration of 1 × 107A/mL, total amount are 1 × 106A/kg；
S3：Cell transplantation, for openheart surgery under extracorporal circulatory system, in cardiac arrest, with myocardium protecting liquid, perfusion
To coronary artery, it to be used for the patient of acute coronary artery syndrome, when percutaneous percutaneous transluminal coronary stent implantation, is noted through conduit after stenter to implant
It is incident upon coronary artery.
Further, the adjustment type macrophage induced through above-mentioned condition can be used for openheart surgery and urgency through the above method
The patient of property coronary syndrome.
Through sufficient zoopery, as a result prove that the cell therapy can effectively mitigate Myocardial Ischemia-Reperfusion,
Mitigate myocardial fibrosis at a specified future date, improves heart function.Flow diagram such as Fig. 1.
Specifically it act as：One, chmice acute ischemia-reperfusion injury model (Reperfu- sion 2h after ligation descending anterior branch 30min) is established,
Transexocardial injects the cell when myocardial ischemia, discovery can its substantially reduced ischemical reperfusion injury, including Troponin I (figure
3), infarct size (Fig. 4), cardiac muscle cell apoptosis (Fig. 5)；Two, rat chronic myocardial ischemia-reperfusion model is established (to drop before ligation
Reperfu- sion 2 weeks after branch 45min), transexocardial injects the cell when myocardial ischemia, after finding 2 weeks its myocardial fibrosis obviously subtract
Gently (Fig. 6), heart function are obviously improved (Fig. 7).Therefore：Sham-operation group, CK：Model untreated group, MT：Model+cell therapy
Group.
Therefore we demonstrate that the cell to myocardial ischemia-reperfusion injury, including it is acute and chronic all have protective effect.It can
Be further used in clinic, improve openheart surgery after Myocardial protective effects, improve patients ' life quality, be one take into account it is close, remote
The myocardial preservation measure that phase influences.
It although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
Understanding without departing from the principles and spirit of the present invention can carry out these embodiments a variety of variations, modification, replace
And modification, the scope of the present invention is defined by the appended.
Claims (2)
1. a kind of method that the adjustment type macrophage of in vitro culture induction is used for myocardial preservation, it is characterised in that：This method packet
Include following steps：
S1：Extract derived from bone marrow macrophage, culture induction adjustment type macrophage, it is all operation under aseptic condition into
Row, concrete operations are as follows：
One, bone marrow aspiration takes autologous patient marrow, external to be resuspended into RPMI-1640 culture mediums, the filtering of 200 mesh filter screens, 500g
5min is centrifuged, supernatant is abandoned, cell is resuspended to BMM culture mediums in erythrocyte cracked liquid splitting erythrocyte；
Two, cell is placed in 37 DEG C, 50mL/L CO2 incubators, per changing within 2-3 days liquid (fresh M-CSF need to be added by changing liquid every time), 7 days at
For ripe monocytes/macrophages (M0)；
Three, the M0 of induced maturation stimulates 48h as follows：Chicken egg white (OVA)-lgG immune complexs (15 μ g/ml OVA and 150
μ g/ml anti ova-lgG, 37 DEG C of incubation 30min)+LPS (50ng/ml), i.e. induction is ripe adjustment type macrophage；
S2：Prepare macrophage suspension：Adjustment type macrophage is resuspended using pancreatin digestion into physiological saline using preceding, adjustment
A concentration of 1 × 107A/mL, total amount are 1 × 106A/kg；
S3：Cell transplantation, for openheart surgery under extracorporal circulatory system, in cardiac arrest, with myocardium protecting liquid, perfusion to hat
Shape artery is used for the patient of acute coronary artery syndrome, when percutaneous percutaneous transluminal coronary stent implantation, is injected to through conduit after stenter to implant
Coronary artery.
2. the method that a kind of adjustment type macrophage of in vitro culture induction according to claim 1 is used for myocardial preservation,
It is characterized in that：The adjustment type macrophage induced through above-mentioned condition can be used for openheart surgery and acute coronary through the above method
The patient of syndrome.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201810200942.2A CN108396009A (en) | 2018-03-12 | 2018-03-12 | A kind of method that the adjustment type macrophage of in vitro culture induction is used for myocardial preservation |
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| CN201810200942.2A CN108396009A (en) | 2018-03-12 | 2018-03-12 | A kind of method that the adjustment type macrophage of in vitro culture induction is used for myocardial preservation |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112716913A (en) * | 2020-12-31 | 2021-04-30 | 上海市胸科医院 | Bionic nano-drug targeting myocardial infarction part and preparation method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1533431A (en) * | 2001-04-13 | 2004-09-29 | Methods and reagents for cell transplantation | |
| CN102732482A (en) * | 2012-05-14 | 2012-10-17 | 中国人民解放军第三军医大学 | In vitro induction culture method for bone marrow-derived macrophages |
-
2018
- 2018-03-12 CN CN201810200942.2A patent/CN108396009A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1533431A (en) * | 2001-04-13 | 2004-09-29 | Methods and reagents for cell transplantation | |
| CN102732482A (en) * | 2012-05-14 | 2012-10-17 | 中国人民解放军第三军医大学 | In vitro induction culture method for bone marrow-derived macrophages |
Non-Patent Citations (2)
| Title |
|---|
| YUANYUE等: "M2b macrophages reduce early reperfusion injury after myocardial ischemia in mice: A predominant role of inhibiting apoptosis via A20", 《INTERNATIONAL JOURNAL OF CARDIOLOGY》 * |
| 宛硕等: "巨噬细胞极化的研究进展", 《中国病原生物学杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112716913A (en) * | 2020-12-31 | 2021-04-30 | 上海市胸科医院 | Bionic nano-drug targeting myocardial infarction part and preparation method thereof |
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Application publication date: 20180814 |