CN102008507A - Human umbilical cord mesenchymal stem cell (HUMSC) anti-hepatic fibrosis injection and preparation method thereof - Google Patents
Human umbilical cord mesenchymal stem cell (HUMSC) anti-hepatic fibrosis injection and preparation method thereof Download PDFInfo
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Images
Abstract
The invention discloses a human umbilical cord mesenchymal stem cell (HUMSC) anti-hepatic fibrosis injection and a preparation method thereof. The injection is composed of 10%-50% of human albumin stock solution, 49.5%-89.5% of acetated Ringer's solution and 0.5% of heparin calcium, wherein, the concentration of the human albumin stock solution is 10%, and 1ml of the injection contains 6*10<5>-7*10<5> HUMSCs. The preparation method comprises the following steps: providing an MSC preserving fluid and pre-cooling the MSC preserving fluid for later use, wherein, the MSC preserving fluid comprises the human albumin stock solution, the Acetated Ringer's solution and the heparin calcium; providing the HUMSCs and adding the HUMSCs to the MSC preserving fluid; and adjusting the number of the HUMSCs added to the MSC preserving fluid to be 6*10<5>-7*10<5> HUMSCs/ml by means of re-suspending. The injection provided by the invention has the advantages of obvious curative effect and high stability, and is convenient in storage and transport and safe in use, thus being capable of bringing gospel to patients with hepatic fibrosis.
Description
[technical field]
The present invention relates to the preparation method of injection, relate in particular to a kind of human umbilical cord mesenchymal stem cells anti-hepatic fibrosis injection and preparation method thereof.
[background technology]
Hepatic fibrosis is to be secondary to the liver inflammation that a variety of causes causes or the compensation response of damage back process of tissue reparation, is the result of extracellular matrix sedimentary pathological change of transition in liver, also is the important intermediate link that chronic hepatopathy develops into liver cirrhosis.The hepatic lesions in this stage is reversible at present, but if do not treated timely and effectively, then finally often cause liver cirrhosis, upper gastrointestinal hemorrhage, hepatic ascites, hepatocarcinoma and liver failure etc., be to cause one of human main causes of death, thereby reverse or delay the important step that liver fibrosis process is a liver disease.The cause of disease that causes hepatic fibrosis is a lot, and at home with viral hepatitis, especially the hepatic fibrosis due to chronic type b and the hepatitis C is the most common.In China, the recall rate of hepatitis B virus surface antigen is about 10%, and wherein chronic asymptomatic hepatitis b virus infected person or chronic asymptomatic HBV carrier just surpass 1.3 hundred million, existing about 3,000 ten thousand people of chronic viral hepatitis B patient.40% liver cirrhosis or hepatocarcinoma may take place in the chronic infectious patients, are 100-200 times of normal population.Abroad, particularly North America, West Europe then are maximum with the alcoholic fibrosis.Hepatic fibrosis can appear in some liver congestion, cholestasis, autoimmune liver disease hereditary and that metabolic disease, chemical toxicant or drug induced injury, schistosomicide, a variety of causes cause in addition.
Therefore, the treatment of hepatic fibrosis becomes the focus of people's concern now.How preventing and treating hepatic fibrosis effectively, block its progress, the prognosis that improves chronic hepatopathy is had important meaning, not only is that country saves a large amount of resources, has also promoted society's harmonious development more.Yet modern medicine there is no the specific drug of treatment hepatic fibrosis, the symptomatic treatment modes such as rest, reinforcement nutrition, vitimin supplement that adopt more, and severe patient need be forced to discontinue medication, in order to avoid increase the weight of hepatic fibrosis.Although Chinese medicine has certain short regeneration and repair to hepatic fibrosis, effect still is not very good.
The method treatment hepatic fibrosis of the stem cell transplantation of utilizing is also arranged at present, but adopt mesenchymal stem cells MSCs more, the cell source is limited.And the cell processing procedure is fairly simple before the infusion, can not guarantee the stability of cell in the use, can not the long period keep cytoactive, therefore is unsuitable for remote transportation.And the multiple adhesion molecule of mescenchymal stem cell surface expression mainly participates in cell-cell, and the specificity adhesion process between the cell-matrix influences cell migration.Therefore, in the stem cell infusion process, very easily impel cell aggregation agglomerating, form the cell bolt, increased the danger of mescenchymal stem cell clinical treatment.To this problem, still there is not fabulous solution at present.
The mescenchymal stem cell that the present invention adopts human umbilical tissue to separate adds calciparine, human albumin, vigorous arteries and veins power A, is prepared into the injection form.Stability is high, preferably resolves the agglomerating problem of cell aggregation, has increased injection preservation and transportation stability, has improved clinical safety in utilization, and then better kept the curative effect of injection, for clinical use stem cell has been opened up new road.
[summary of the invention]
The objective of the invention is in order to overcome the deficiencies in the prior art, a kind of human umbilical cord mesenchymal stem cells anti-hepatic fibrosis injection and preparation method thereof is provided, its have stability height, good effect, safe, be convenient to store and transport, adapt to clinical extensive use, the characteristics that have a extensive future, the present invention can be patients with liver fibrosis and brings glad tidings, for new road has been opened up in the use of stem cell clinically.
Human umbilical cord mesenchymal stem cells anti-hepatic fibrosis injection provided by the invention, vigorous arteries and veins power A liquid and 0.5% calciparine by 10%-50% human albumin stock solution, 49.5%89.50% are formed, the concentration of wherein said human albumin's stock solution is 10%, contains human umbilical cord mesenchymal stem cells 6 * 10 in the 1ml injection
5-7 * 10
5Individual.Calciparine is a kind of polysaccharide compound that contains, and has the preparation that prevents the agglomerating function of cell aggregation.
Described injection is made up of 20% human albumin's stock solution and 79.5% vigorous arteries and veins power A liquid and 0.5% calciparine, and wherein the concentration of the human umbilical cord mesenchymal stem cells in this injection is 6.8 * 10
5Individual/ml.
Described injection uses the venoclysis mode, according to human umbilical cord mesenchymal stem cells 0.5 * 10
6-1 * 10
6Individual cell/kg body weight is injected in patient's body.
The preparation method of the human umbilical cord mesenchymal stem cells anti-hepatic fibrosis injection that the present invention provides again, comprise: mesenchyme stem cell preserving fluid is provided, pre-cooling, standby, described mesenchyme stem cell preserving fluid comprise human albumin's stock solution, vigorous arteries and veins power A liquid and calciparine; Human umbilical cord mesenchymal stem cells is provided; The human umbilical cord mesenchymal stem cells that obtains is joined in the mesenchyme stem cell preserving fluid; By resuspended mode, adjusting the human umbilical cord mesenchymal stem cells quantity that joins in the mesenchyme stem cell preserving fluid is 6 * 10
5-7 * 10
5Individual cell/1ml.
Described mesenchyme stem cell preserving fluid is that 10% the vigorous arteries and veins power of human albumin's stock solution 49.50%-89.5% A liquid, 0.5% calciparine mixing are formed by the concentration of 10%-50%.
Described mesenchyme stem cell preserving fluid is made up of 20% human albumin's stock solution and 79.5% vigorous arteries and veins power A liquid, 0.5% calciparine.
Described human umbilical cord mesenchymal stem cells prepares by following mode: (1) picks and places the culture bottle of 3-7 for human umbilical cord mesenchymal stem cells; (3) original culture medium in the culture bottle is abandoned in suction, and every bottle of D-hank ' s balanced salt solution buffer solution for cleaning twice with 10ml is inhaled and abandoned washing liquid; (4) add the 1ml pancreas enzyme-EDTA in every bottle, put into 37 ℃ of incubator 5-7 minutes; (5) observation of cell under inverted microscope is patted culture bottle gently with palm and is guaranteed that the human umbilical cord mesenchymal stem cells catapepsis gets off; (6) add 0.2ml serum in every bottle and stop digestion; Add D-hank ' the s balanced salt solution of 10ml in every bottle, piping and druming makes cell suspension repeatedly; (7) cell suspension is moved in the centrifuge tube, with the centrifugal speed of 900rpm centrifugal 5 seconds, centrifugal end was inhaled and is abandoned supernatant, obtains human umbilical cord mesenchymal stem cells.
Adjust cell concentration in the stem cell preserving fluid by following mode: (8) are with the resuspended human umbilical cord mesenchymal stem cells of mesenchyme stem cell preserving fluid, draw a little cell suspension with the micro sample-adding rifle, be used as cell counting and cell viability and detect, give up dead cell, and living cell counting; As viable count concentration is 6 * 10
5-7 * 10
5Individual cell/1ml carries out step (10); Otherwise continue to adjust again with step (9); (9) then centrifugal 5 seconds with the centrifugal speed of 900rpm, inhale and abandon supernatant, add and preserve liquid, making cell concentration is 6 * 10
5-7 * 10
5Individual cell/1ml carries out step (10) again; (10) with adjusted 6 * 10
5-7 * 10
5The human umbilical cord mesenchymal stem cells anti-hepatic fibrosis injection of individual cell/1ml encapsulates preservation.
Suction is abandoned to be needed before original culture medium in the culture bottle the culture bottle disinfection, is specially with around the cotton ball soaked in alcohol wiping cultivation bottle cap mouth bottle cap of outwarding winding then.
Described cell counting and cell viability detect and are specially: the platform of cell suspension and 0.4% is expected that blue solution mixed by 1: 1; Mixing gently is after 1 minute, at microscopically blood counting chamber counting cells; Living cells repels platform and expects orchid, and dying blue cell is dead cell.
The present invention compared with prior art has the following advantages:
The present invention has broken the traditional therapy of hepatic fibrosis, use the mescenchymal stem cell in people's umbilical cord source (to be called for short: MSCs cleverly, English full name: mesenchymal stem cells), and it is become the injection form with other composition combined preparation, it is human umbilical cord mesenchymal stem cells anti-hepatic fibrosis injection, its stability is high, solve prior art cell in the stem cell infusion process and assembled agglomerating problem easily, and prolonged guaranteeing the quality the time of cell to be used, 24h inner cell motility rate still maintains more than 90%, good effect, use safer, be suitable for preservation and the transportation of long period, without any side effects with the back, for clinical extensive use is laid a good foundation.Injection provided by the invention can be divided into the hepatocyte of function; Have Regeneration and Repair organa parenchymatosum and the immunoregulatory two-way function of performance, improve immunologic function, improve the microenvironment of liver and reduce the excretory ability of inflammatory cytokine, reaction reduces inflammation; Suppress the generation of hepatic stellate cell activation, prevention extracellular matrix, reverse hepatic fibrosis; Activate oogonium, promote liver cell regeneration, repair organa parenchymatosum and chronic hepatic injury, improved the antiviral therapy curative effect indirectly.And the human umbilical cord mesenchymal stem cells in the injection of the present invention is not because wide material sources are hanged down by ethics restriction, immunogenicity and make it become the new effective means for the treatment of liver fibrosis, and application prospect is very wide.
[description of drawings]
Fig. 1 is the flow chart of the preparation method of human umbilical cord mesenchymal stem cells anti-hepatic fibrosis injection provided by the invention.
Fig. 2 is the photo of human umbilical cord mesenchymal stem cells anti-hepatic fibrosis injection provided by the invention with microscopically after cell solution identical with human umbilical cord mesenchymal stem cells anti-hepatic fibrosis injectable liquefied composition provided by the invention but that do not add calciparine leaves standstill 12h.Wherein A, B are that human umbilical cord mesenchymal stem cells anti-hepatic fibrosis injection provided by the invention leaves standstill microscopically photo behind the 12h, and wherein A is 4 times of photos under the mirror, and B is 10 times of photos under the mirror; C, D contain cell solution identical with human umbilical cord mesenchymal stem cells anti-hepatic fibrosis injectable liquefied composition provided by the invention but that do not add calciparine to leave standstill 12h microscopically photo, and wherein C is 4 times of photos under the mirror, and D is 10 times of photos under the mirror.
[specific embodiment]
Reach technological means and the effect that predetermined purpose is taked for further setting forth the present invention, below in conjunction with accompanying drawing and preferred embodiment, human umbilical cord mesenchymal stem cells anti-hepatic fibrosis injection that foundation the present invention is proposed and preparation method thereof, its specific embodiment, structure, feature and effect thereof, illustrate as after.
Human umbilical cord mesenchymal stem cells anti-hepatic fibrosis injection provided by the invention, form by the human albumin's stock solution of 10%-50% and vigorous arteries and veins power A liquid, 0.5% calciparine of 49.5%-89.5%, the concentration of described human albumin's stock solution is 10%, and the concentration of human umbilical cord mesenchymal stem cells is 6 * 10 in the injection
5-7 * 10
5Individual/ml.The injection of the embodiment of the invention is that 10% human albumin's stock solution and 79.5% vigorous arteries and veins power A liquid, 0.5% calciparine are formed by 20% concentration, and wherein the concentration of the human umbilical cord mesenchymal stem cells in this injection is 6.8 * 10
5Individual/ml.Described injection uses the venoclysis mode, according to human umbilical cord mesenchymal stem cells 0.5 * 10
6-1 * 10
6Individual cell/kg body weight is injected in patient's body.Human albumin's stock solution in the injection of the present invention is that the accurate word of traditional Chinese medicines that Haerbin Shiheng Bioengineering Medicine Stock Co., Ltd. produces is human albumin's stock solution of S10920005, and this original liquid concentration is 10%.
The preparation method of human umbilical cord mesenchymal stem cells anti-hepatic fibrosis injection provided by the invention as shown in Figure 1, comprises four steps:
1, provide mesenchyme stem cell preserving fluid, pre-cooling, standby, described mesenchyme stem cell preserving fluid comprises human albumin's stock solution, vigorous arteries and veins power A liquid, calciparine;
2, provide human umbilical cord mesenchymal stem cells;
3, the human umbilical cord mesenchymal stem cells that obtains is joined in the mesenchyme stem cell preserving fluid;
4, by resuspended mode, adjusting the human umbilical cord mesenchymal stem cells quantity that joins in the mesenchyme stem cell preserving fluid is 6 * 10
5-7 * 10
5Individual cell/1ml.
The mesenchyme stem cell preserving fluid of the embodiment of the invention is that 10% human albumin's stock solution and the vigorous arteries and veins power of 49.5%-89.5% A liquid, 0.5% calciparine mixing are formed by the concentration of 10%-50%.
The preparation method of the human umbilical cord mesenchymal stem cells anti-hepatic fibrosis injection that the embodiment of the invention provides specifically may further comprise the steps:
1, provide mesenchyme stem cell preserving fluid, pre-cooling, standby, this mesenchyme stem cell preserving fluid is made up of 20% human albumin's stock solution and 79.5% vigorous arteries and veins power A liquid, 0.5% calciparine, and wherein the concentration of this human albumin's stock solution is 10%.
2, by following step (1) to (7) preparation and the required human umbilical cord mesenchymal stem cells of acquisition injection of the present invention:
(1) picks and places the culture bottle of 3-7 for human umbilical cord mesenchymal stem cells;
(2) with around the cotton ball soaked in alcohol wiping cultivation bottle cap mouth, the bottle cap of outwarding winding then;
(3) original culture medium in the culture bottle is abandoned in suction, and (this D-hank ' s balanced salt solution buffer contains 0.8% NaCl, 0.04% KCl, 0.006% Na to every bottle of D-hank ' s balanced salt solution buffer with 10ml
2HPO
4H
2O and 0.006% KH
2PO
4) clean twice, inhale and abandon washing liquid;
(4) add 1ml pancreas enzyme-EDTA (the Chinese full name of EDTA: sodium ethylene diamine tetracetate, English full name: ethylenediamine traacetic acid), put into 37 ℃ of incubator 5-7 minutes in every bottle;
(5) observation of cell under inverted microscope is patted culture bottle gently with palm and is guaranteed that the human umbilical cord mesenchymal stem cells catapepsis gets off;
(6) add 0.2ml serum in every bottle and stop digestion; Add D-hank ' the s balanced salt solution of 10ml in every bottle, piping and druming makes cell suspension repeatedly;
(7) cell suspension is moved in the centrifuge tube, with the centrifugal speed of 900rpm centrifugal 5 seconds, centrifugal end was inhaled and is abandoned supernatant, obtains human umbilical cord mesenchymal stem cells.
3, the human umbilical cord mesenchymal stem cells that obtains is joined in the mesenchyme stem cell preserving fluid.
4, by resuspended mode, adjusting the human umbilical cord mesenchymal stem cells quantity that joins in the mesenchyme stem cell preserving fluid is 6 * 10
5-7 * 10
5Individual cell/1ml, concrete adjustment process is:
(8) with the resuspended human umbilical cord mesenchymal stem cells of mesenchyme stem cell preserving fluid, draw a little cell suspension, be used as cell counting and cell viability and detect, give up dead cell with the micro sample-adding rifle, and living cell counting; As viable count concentration is 6 * 10
5-7 * 10
5Individual cell/1ml carries out step (10); Otherwise continue to adjust again with step (9); Described cell counting and cell viability detect and are specially: the platform of cell suspension and 0.4% is expected that blue solution mixed by 1: 1; Mixing gently is after 1 minute, at microscopically blood counting chamber counting cells; Living cells repels platform and expects orchid, and dying blue cell is dead cell;
(9) then centrifugal 5 seconds with the centrifugal speed of 900rpm, inhale and abandon supernatant, add and preserve liquid, making cell concentration is 6 * 10
5-7 * 10
5Individual cell/1ml carries out step (10) again;
(10) with adjusted 6 * 10
5-7 * 10
5The human umbilical cord mesenchymal stem cells anti-hepatic fibrosis injection of individual cell/1ml encapsulates preservation.
Be 50ml with the mesenchyme stem cell preserving fluid below, making human umbilical cord mesenchymal stem cells concentration is 6.8 * 10
5Individual cell/1ml is an example, carries out the preparation process of concrete detailed explanation injection of the present invention:
(1), draw human albumin's stock solution 10ml, the 0.25ml calciparine, remainder is supplemented to 50ml with vigorous arteries and veins power A liquid, mixing makes mesenchyme stem cell preserving fluid, pre-cooling, standby;
(2), get the culture bottle of 3-7 for human umbilical cord mesenchymal stem cells is housed;
(3), use the cotton ball soaked in alcohol wiping to cultivate around the bottle cap mouth bottle cap of outwarding winding;
(4), inhale and to abandon original culture medium in the culture bottle, every bottle of D-hank ' s buffer solution for cleaning twice with 10ml is inhaled and is abandoned washing liquid;
(5), add the 1ml pancreas enzyme-EDTA in every bottle, put into 37 ℃ of incubator 5-7 minutes;
(6), under inverted microscope observation of cell, pat culture bottle gently with palm and guarantee that the human umbilical cord mesenchymal stem cells catapepsis gets off;
(7), add 0.2ml serum in every bottle and stop digestion;
(8), add D-hank ' the s buffer of 10ml in every bottle, piping and druming makes cell suspension repeatedly;
(9), cell suspension is moved in some centrifuge tubes, with the centrifugal speed of 900rpm centrifugal 5 seconds, centrifugal end was inhaled and is abandoned supernatant, makes human umbilical cord mesenchymal stem cells;
(10), take out required mesenchyme stem cell preserving fluid, the human umbilical cord mesenchymal stem cells that obtains is joined in the mesenchyme stem cell preserving fluid;
(11), with 20ml mesenchyme stem cell preserving fluid resuspended human umbilical cord mesenchymal stem cells, and merge in the 50ml centrifuge tube;
(12), draw a little cell suspension, be used as cell counting and cell viability and detect, give up dead cell with the micro sample-adding rifle, and living cell counting; Described cell counting and cell viability detect and are specially: the platform of cell suspension and 0.4% is expected that blue solution mixed by 1: 1; Mixing gently, after 1 minute, at microscopically blood counting chamber counting cells, living cells repels platform and expects orchid, thereby to dye blue cell be dead cell;
(13), be 6.8 * 10 as viable count concentration
5Individual cell/1ml carries out step (15); Otherwise continue to adjust again with step (14);
(14), then with the centrifugal 5min of the centrifugal speed of 900rpm, inhale and to abandon supernatant, add and preserve liquid, making cell concentration is 6.8 * 10
5Individual cell/1ml carries out step (15) again;
(15), with adjusted 6.8 * 10
5The human umbilical cord mesenchymal stem cells anti-hepatic fibrosis injection of individual cell/1ml encapsulates preservation.
The human umbilical cord mesenchymal stem cells anti-hepatic fibrosis injection for preparing need be under 4-8 ℃ of lucifuge environment storage and transport, effect duration is 12 hours in the packing.Injection of the present invention is applicable to venoclysis, calculates by weight in patients kilogram number, and recommending consumption is 0.5 * 10
6-1 * 10
6Individual cell/kg body weight.
With injection provided by the invention and do not contain calciparine injection the two all place under 4 ℃ the temperature environment and preserve.After preserving 24 hours, take a sample, and examine under a microscope the gathering situation of cell in two kinds of injections.As shown in Figure 2, under equal preservation condition, injection provided by the invention has good stability at the 24h inner cell, does not assemble agglomerating phenomenon, and that the cell solution that does not add anti-aggregating agent prepared therefrom is assembled agglomerating phenomenon is serious.Therefore, add anti-aggregating agent prepared therefrom and improved cell solution stability greatly, reduced clinical application risk.
By four parts of human umbilical cord mesenchymal stem cells anti-hepatic fibrosis injection that contain the variable concentrations human albumin of the method for the invention preparation, wherein human albumin's stock solution additional proportion is respectively 5%, 10%, 20%, 50%, the concentration of used human albumin's stock solution is 10%, vigorous arteries and veins power A content is respectively 94.5%, 89.5%, 79.5%, 49.5%, calciparine content is 0.5%, and the concentration of the human umbilical cord mesenchymal stem cells in the injection is 6.8 * 10
5Individual/ml.Above four duplicate samples that prepare are placed under 4 ℃ of temperature preserve, sampling when preserving 12 hours, and measure cell survival rate.
Assay method: cell suspension 0.5ml is added in the test tube; Add 0.5ml concentration and be 0.2% platform and expect blue dye liquor; With cell suspension sucking-off 10 μ l, drip at the cover plate edge, suspension is full of between cover plate and the counting chamber; Left standstill 1 minute; The several visuals field of picked at random amount to several 300 cells, and count wherein dead cell number, the meter cell viability, and repeat count is averaged for 5 times.Shown in form 1-3, be the test of selecting for use three different umbilical cords to carry out.But 0.05 the p value be considered to the border level of acceptance error usually.
Form 1 (P＞0.05)
Form 2 (P＞0.05)
Form 3 (P＞0.05)
The result shows, the former liquid proportional of human albumin in preserving liquid is greater than 10% the time, cell survival rate is all higher, therefore, we select the preservation liquid of being made umbilical cord mesenchymal stem cells by human albumin's stock solution of 20% and vigorous arteries and veins power A, calciparine for use, can obviously prolong the cell survival time, keep cytoactive.
The resuspended ordinary cells solution of vigorous arteries and veins power A that injection of the present invention is identical with cell concentration is put in 4 ℃ of preservations, respectively at the time point of preserving 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 24 hours, sampling and measuring cell survival rate.Shown in form 4-6, for selecting three different batches human umbilical cord mesenchymal stem cells anti-hepatic fibrosis injection repeated trials for use.
The concrete method of measuring cell survival rate is with blood counting chamber and cover plate wiped clean, and cover plate is covered on counting chamber; The cell suspension of 0.5ml is added in the test tube; Add the 1.5ml0.2% platform and expect blue dye liquor; With cell suspension sucking-off 10 μ l, drip at the cover plate edge, suspension is full of between cover plate and the counting chamber; Left standstill 1 minute; The several visuals field of picked at random amount to several 300 cells, and count wherein dead cell number, the meter cell viability, and repeat count is got mean 5 times.
Form 4 (P＞0.05)
Form 5 (P＞0.05)
Form 6 (P＞0.05)
The result is presented under the same preservation liquid condition, and injection group preservation effect obviously is better than the preservation effect of common group (solution that promptly has only vigorous arteries and veins power A liquid and cell); Under 4 ℃ of conditions, preserve 24 hours survival rates and can maintain more than 90%; Therefore human umbilical cord mesenchymal stem cells anti-hepatic fibrosis injection of the present invention can obviously prolong the cell survival time, has strengthened cell stability, is convenient to preserve and clinical transportation use.
Use the patient of injection provided by the invention all to be selected from the inpatient, 10 examples altogether, hbv-liver cirrhosis 9 examples wherein, autoimmunity liver cirrhosis 1 example is and loses the compensatory phase.After importing injection of the present invention, patient's spirit takes a turn for the better, and appetite increases, and ascites disappears, and the serology biochemical indicator obviously improves, and after the treatment, symptom and sign obviously improves, and reaches the requirement of leaving hospital for the first time.The hbv-liver cirrhosis patient takes antiviral drugs routinely.
Following table is learned index (meansigma methods) for the patients serum.(ALT (alanine aminotransferase): alanine aminotransferase; AST (aspartate aminotransferase): glutamic oxaloacetic transaminase, GOT; ALB (albumin): serum albumin; TBIL (total bilirubin): total bilirubin; PT (prothrombintime): prothrombin time; PT%: prothrombin time mobility; PTINR (prothrombintime internationalnormalized ratio): prothrombin time international normalized ratio; AFP (alpha-fetal protein): alpha-fetoprotein)
| ALT | AST | ALB | TBIL | PT | PT％ | PT?INR | AFP | |
| Before the treatment | 79.7 | 119.96 | 25.97 | 41.58 | 15.48 | 78.33 | 1.16 | 2.7 |
| After the treatment | 38.84 | 59.97 | 35.27 | 2.13 | 14.13 | 88.07 | 1.07 | 2.2 |
All patients use human umbilical cord mesenchymal stem cells anti-hepatic fibrosis injection for treating 3 times, are separated by one-month period at every turn, and patient is after treatment, and liver function injury alleviates, and liver cell regeneration is active, and albumin secretion increases, and coagulation function recovers normally.For the treatment safety of clear and definite human umbilical cord mesenchymal stem cells anti-hepatic fibrosis injection, every routine patient, all level of routine examination AFP.After treatment, the level of patient AFP is no significant difference before treatment and after the treatment, even slight attenuating is arranged.
The mescenchymal stem cell that the present invention adopts human umbilical tissue to separate, it is different from bone marrow and Cord blood mescenchymal stem cell, and it is more extensive to originate, and add calciparine, human albumin, vigorous arteries and veins power A, be prepared into the injection form, preferably resolve the agglomerating problem of cell aggregation, increased injection preservation and transportation stability, clinical safety in utilization, and then better kept the curative effect of injection, be convenient to clinical extensive use.
The present invention extracts mescenchymal stem cell by the umbilical cord that utilizes the full-term pregnancy fetus (to be called for short: MSCs, English full name: mesenchymal stem cells), preparation can be for the human umbilical cord mesenchymal stem cells anti-hepatic fibrosis injection of clinical use, its stability is high, be convenient to store and transportation, evident in efficacy, safe, without any side effects with the back, be suitable for clinical extensive use.Injection provided by the invention can be divided into the hepatocyte of function, have Regeneration and Repair organa parenchymatosum and the immunoregulatory two-way function of performance, improve immunologic function, improve the microenvironment of liver and reduce the excretory ability of inflammatory cytokine, reaction reduces inflammation; Suppress the generation of hepatic stellate cell activation, prevention extracellular matrix, reverse hepatic fibrosis; Activate oogonium, promote liver cell regeneration, repair organa parenchymatosum and chronic hepatic injury, improved the antiviral therapy curative effect indirectly.And the human umbilical cord mesenchymal stem cells in the injection of the present invention is not because wide material sources are hanged down by ethics restriction, immunogenicity and make it become the new effective means for the treatment of liver fibrosis, and application prospect is very wide.
In this description, the present invention is described with reference to its certain embodiments, still, still can make various modifications and conversion obviously and does not deviate from the spirit and scope of the present invention.Therefore, description of the present invention and accompanying drawing are considered to illustrative and nonrestrictive.
Claims (10)
1. human umbilical cord mesenchymal stem cells anti-hepatic fibrosis injection, it is characterized in that, vigorous arteries and veins power A liquid, 0.5% calciparine by 10%-50% human albumin stock solution and 49.5%-89.5% are formed, the concentration of wherein said human albumin's stock solution is 10%, contains human umbilical cord mesenchymal stem cells 6 * 10 in the 1ml injection
5-7 * 10
5Individual.
2. human umbilical cord mesenchymal stem cells anti-hepatic fibrosis injection as claimed in claim 1, it is characterized in that, described injection is made up of 20% human albumin's stock solution and 79.5% vigorous arteries and veins power A liquid, 0.5% calciparine, and wherein the concentration of the human umbilical cord mesenchymal stem cells in this injection is 6.8 * 10
5Individual/ml.
3. human umbilical cord mesenchymal stem cells anti-hepatic fibrosis injection as claimed in claim 2 is characterized in that, described injection uses the venoclysis mode, according to human umbilical cord mesenchymal stem cells 0.5 * 10
6-1 * 10
6Individual cell/kg body weight is injected in patient's body.
4. the preparation method of the described human umbilical cord mesenchymal stem cells anti-hepatic fibrosis of claim 1 injection is characterized in that, comprising:
Mesenchyme stem cell preserving fluid is provided, pre-cooling, standby, described mesenchyme stem cell preserving fluid comprises human albumin's stock solution, vigorous arteries and veins power A liquid and calciparine;
Human umbilical cord mesenchymal stem cells is provided;
The human umbilical cord mesenchymal stem cells that obtains is joined in the mesenchyme stem cell preserving fluid;
By resuspended mode, adjusting the human umbilical cord mesenchymal stem cells quantity that joins in the mesenchyme stem cell preserving fluid is 6 * 10
5-7 * 10
5Individual cell/1ml.
5. the preparation method of human umbilical cord mesenchymal stem cells anti-hepatic fibrosis injection as claimed in claim 4, it is characterized in that described mesenchyme stem cell preserving fluid is that 10% human albumin's stock solution and the vigorous arteries and veins power of 49.5%-89.5% A liquid, 0.5% calciparine mixing are formed by the concentration of 10%-50%.
6. the preparation method of human umbilical cord mesenchymal stem cells anti-hepatic fibrosis injection as claimed in claim 5 is characterized in that, described mesenchyme stem cell preserving fluid is made up of 20% human albumin's stock solution and 79.5% vigorous arteries and veins power A liquid, 0.5% calciparine.
7. the preparation method of human umbilical cord mesenchymal stem cells anti-hepatic fibrosis injection as claimed in claim 6 is characterized in that, described human umbilical cord mesenchymal stem cells prepares by following mode:
(1) picks and places the culture bottle of 3-7 for human umbilical cord mesenchymal stem cells;
(3) original culture medium in the culture bottle is abandoned in suction, and every bottle of D-hank ' s balanced salt solution buffer solution for cleaning twice with 10ml is inhaled and abandoned washing liquid;
(4) add the 1ml pancreas enzyme-EDTA in every bottle, put into 37 ℃ of incubator 5-7 minutes;
(5) observation of cell under inverted microscope is patted culture bottle gently with palm and is guaranteed that the human umbilical cord mesenchymal stem cells catapepsis gets off;
(6) add 0.2ml serum in every bottle and stop digestion; Add D-hank ' the s balanced salt solution of 10ml in every bottle, piping and druming makes cell suspension repeatedly;
(7) cell suspension is moved in the centrifuge tube, with the centrifugal speed of 900rpm centrifugal 5 seconds, centrifugal end was inhaled and is abandoned supernatant, obtains human umbilical cord mesenchymal stem cells.
8. the preparation method of human umbilical cord mesenchymal stem cells anti-hepatic fibrosis injection as claimed in claim 7 is characterized in that, adjusts cell concentration in the stem cell preserving fluid by following mode:
(8) with the resuspended human umbilical cord mesenchymal stem cells of mesenchyme stem cell preserving fluid, draw a little cell suspension, be used as cell counting and cell viability and detect, give up dead cell with the micro sample-adding rifle, and living cell counting; As viable count concentration is 6 * 10
5-7 * 10
5Individual cell/1ml carries out step (10); Otherwise continue to adjust again with step (9);
(9) then centrifugal 5 seconds with the centrifugal speed of 900rpm, inhale and abandon supernatant, add and preserve liquid, making cell concentration is 6 * 10
5-7 * 10
5Individual cell/1ml carries out step (10) again;
(10) with adjusted 6 * 10
5-7 * 10
5The human umbilical cord mesenchymal stem cells anti-hepatic fibrosis injection of individual cell/1ml encapsulates preservation.
9. the preparation method of human umbilical cord mesenchymal stem cells anti-hepatic fibrosis injection as claimed in claim 8, it is characterized in that, suction is abandoned to be needed before original culture medium in the culture bottle the culture bottle disinfection, is specially with around the cotton ball soaked in alcohol wiping cultivation bottle cap mouth bottle cap of outwarding winding then.
10. the preparation method of human umbilical cord mesenchymal stem cells anti-hepatic fibrosis injection as claimed in claim 9 is characterized in that, described cell counting and cell viability detect and be specially: the platform of cell suspension and 0.4% is expected that blue solution mixed by 1: 1; Mixing gently is after 1 minute, at microscopically blood counting chamber counting cells; Living cells repels platform and expects orchid, and dying blue cell is dead cell.
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