CN105368777A - Construction method of hepatitis vaccine sensitization dendritic cell inducing specific T cell - Google Patents
Construction method of hepatitis vaccine sensitization dendritic cell inducing specific T cell Download PDFInfo
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- CN105368777A CN105368777A CN201510705644.5A CN201510705644A CN105368777A CN 105368777 A CN105368777 A CN 105368777A CN 201510705644 A CN201510705644 A CN 201510705644A CN 105368777 A CN105368777 A CN 105368777A
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Abstract
The invention provides a construction method of a hepatitis vaccine sensitization dendritic cell inducing specific T cell. The method comprises the following steps that (1) an antigen load mononuclear cell derivative dendritic cell is obtained; (2) the T cell is amplified; (3) the specific hepatitis virus T cell is obtained. The method can efficiently present viral antigen.
Description
Technical field
The invention belongs to field of pharmaceutical biology, be specifically related to a kind of construction process of hepatitis vaccine primed dendritic shape cell induction specific T-cells.
Background technology
It is still the primary factor that progress occurs for liver cancer and End-stage liver disease that Chronic HBV (hepatitis B virus) infects.Existing medicine is difficult to remove virus, and clinical cure rate is extremely low, and after drug withdrawal, recurrence rate is high, needs life-long therapy.The core theory of chronic HBV infection is that specific T-cells functional defect causes body to the immunological tolerance of virus.
Dendritic cell (DC) is the key enabler of T cell activation, it is antigen presenting cell the strongest in body, undertake the startup effect of T cell maturation, activation, propagation, and chb patient DC quantity or impaired function make t cell activation be obstructed, cause viral long-term surviving and copy, and recovering DC function to T cell function and have enhancement; Therefore, obtain the DC cell induction specific T-cells of autologous by in-vitro directed cultivation, thus activating cells is immune, the natural immunity and follow-up humoral immunization, carries out target Precise strike, promotes virus sweep, will be expected to breakthrough healing difficulty to HBV in body.Some clinical studyes in recent years use DC vaccine therapy chb to a certain degree can suppress virus, prompting improves angtigen presentation promotion functions T cell and has potentiality and prospect, the factors of comprehensive chb immunological tolerance microenvironment, further investigated transfers angtigen presentation may set foot on chb healing road to T cell influence.
Summary of the invention
An object of the present invention is the technical problem for solving above, provide a kind of and high-level efficiency can offer the construction process of the hepatitis vaccine primed dendritic shape cell induction specific T-cells of virus antigen.
Another object of the present invention is to provide the cell in vitro system utilizing the method obtained.
Another object of the present invention is to provide the outer cell system cell preparation of occlusion body.
Another object of the present invention is to provide the purposes of the method in the cell preparation for the preparation for the treatment of hepatitis.
In order to realize above object, the invention provides a kind of construction process of hepatitis vaccine primed dendritic shape cell induction specific T-cells, it comprises the steps:
(1) antigen load monocyte derived dendritic cell is obtained;
(2) T cell amplification;
(3) C virusspecific T cell is obtained.
Preferably, described step (1) is: gather peripheral blood 10ml, separating peripheral blood mononuclear cells, be resuspended in 4ml AIM ?V nutrient solution, be then placed in 37 DEG C, 50%CO
2adherent culture 2 hours in incubator; Take out after 2 hours, jog, remove suspension cell, attached cell, namely add in monocyte 4mlAIM ?V substratum, this substratum include concentration be 20ng/ml IL ?4 (interleukin-4s) and concentration be 100ng/ml GM ?CSF (Macrophage-Colony stimulating factor); After 3 days, monocyte supplement AIM ?V nutrient solution 2ml, this nutrient solution containing concentration be 20ng/ml IL ?4 and concentration be 100ng/ml GM ?CSF, be still placed in 37 DEG C, 50%CO
2cultivate in incubator, make its induced maturation be monocyte derived dendritic cell (moDCs cell); Add hepatitis virus vaccine stoste 10 μ g to moDCs cell when the 6th day, cultivate 24 hours, obtain antigen load monocyte derived dendritic cell (HPDCs cell).
Preferably, described step (2) is: by the suspension cell obtained in step (1), i.e. T cell, move to containing 4mlAIM ?the T75 culturing bottle of V nutrient solution, with AIM ?V substratum wash 3 times, amount of liquor is 2ml/ time; Then add OKT3 (CD3 monoclonal antibody) that final concentration is 1 μ g/ml and final concentration be 1 μ g/ml Kang ?CD28, be placed in 37 DEG C, 50%CO
2cultivate in incubator; 2nd day: supplement AIM ?V nutrient solution 5 ?10ml in T cell nutrient solution, this nutrient solution included IL ?2 (Bai Jiesu ?2) that concentration is 300IU/ml and mass body volume concentrations is the albumin 1.5ml of 20%; 4th day: supplement AIM ?V nutrient solution 5 ?10ml, this nutrient solution include concentration be 300IU/ml IL ?2 and mass body volume concentrations be the albumin 1.5ml of 20%, continue at 37 DEG C, 50%CO
2be cultured to the 8th day in incubator, obtain the T cell of propagation.
Preferably, described step (3) is: the T cell of the propagation obtained in the antigen load monocyte derived dendritic cell obtained in step (1) and step (2) is mixed Dual culture; 10th day: supplement AIM ?V nutrient solution 5 ?10ml, this nutrient solution include concentration be 300IU/ml IL ?2 and mass body volume concentrations be the albumin 1.5ml of 20%; 12nd day: supplement AIM ?V nutrient solution 5 ?10ml, this nutrient solution include concentration be 300IU/ml IL ?2 and mass body volume concentrations be 20% albumin 1.5ml; 14th day: obtain C virusspecific T cell.Peripheral blood in patients 10ml carries out directional induction DC generation specific T-cells quantity and reaches 10
7?10
8.
More preferably, described hepatitis vaccine is any one in hepatitis A vaccine, Hepatitis B virus vaccine or hepatitis C vaccine.More preferably, described hepatitis vaccine is Hepatitis B virus vaccine.
Present invention also offers the cell in vitro system obtained according to aforesaid method.
Present invention also offers the cell preparation comprising above-mentioned cell in vitro system.
Present invention also offers according to the purposes of aforesaid method in the cell preparation for the preparation for the treatment of hepatitis.
Preferably, described hepatitis is any one in hepatitis A, hepatitis B or the third liver.More preferably, described hepatitis is hepatitis B.
Particularly, the invention discloses the construction process of hepatitis vaccine (particularly Hepatitis B virus vaccine) primed dendritic shape cell (DC) inducing specific T cell (HPDCT), utilize peripheral blood lymphocytes to obtain functional ripe DC by directional induction, then induce CD4+T cell and the CD8+T cell of HBV specificity multi-epitope by adoptive immunity; The responsiveness cytokine IL of described T cell energy secreting high levels ?17, IL ?2, IFN ?γ.
First the present invention prepares the activation DC cell in autologous patient source, utilize peripheral blood in patients PBMC, after separating monocytic cell, cytokine Bai Jiesu ?4 (IL ?4) and G CFS (GSF) induction is utilized to obtain functional DC, with polychrome fluidic cell qualification DC maturation mark; Then to go the Hepatitis B virus vaccine of adjuvant to impact, the DC of surface antigen sensitization is obtained.
T cell amplification is carried out in parallel laboratory test, use OKT3 He Kang ?CD28 set up and optimize Lymphocyte expansion culture system, vitro culture is after 7 days, then with the ripe DC mixed culture of carrier surface antigen, thus obtain HBV specificity IFN ?γ and T cell.
The body outer cell line that present method obtains significantly can improve the specific cellular immunity of patient, obtained cell in vitro system is fed back in patient body, thus target attacks HBV in body, degraded virogene and the infected liver cell of removing, cure for chronic viral hepatitis B and provide new approach.
The relatively difference of combination antiviral medicine superposition HPDCT cell therapy and alone medicine or alone cell therapy T effector cell and cytokine.Found that, antiviral treatment patient monocyte derived Dendritic Cells Induced specific T-cells secretion IFN ?γ level is higher than the chb patient without antiviral therapy.Antiviral superposition HPDCT cell therapy has higher antiviral effect.
Accompanying drawing explanation
Fig. 1 shows the monocyte derived dendritic cell containing ripe mark.
Fig. 2 shows antigen-loaded dendritic cell induced higher levels specific T-cells.
Fig. 3 show antigen-loaded dendritic cell induced higher levels IFN ?γ.
Fig. 4 shows the stronger antiviral effect of antiviral superposition HPDCT display.
Fig. 5 shows ETV single therapy and ETV+HPDCT combination therapy flow process.
Fig. 6 show ETV combine HPDCT treatment group patient produce degraded virus is played a crucial role T cell effector (IL ?17 and IFN ?γ) be significantly higher than ETV single therapy.
Embodiment
Below in conjunction with the drawings and specific embodiments, technical scheme of the present invention is described in further detail, but the present invention is not limited to following examples.Experimental technique in following embodiment, if no special instructions, is ordinary method.The statement of following number of days, as do not particularly not pointed out, all from build time calculate.
Embodiment 1: obtain antigen load monocyte derived dendritic cell
Gather Peripheral Blood in Patients with Chronic Hepatitis B 10ml, conventionally separating peripheral blood mononuclear cells (PBMC), be resuspended in 4ml AIM ?V nutrient solution, be then placed in 37 DEG C, 50% (volumetric concentration) CO
2adherent culture 2 hours in incubator.
After 2 hours take out, jog, removes the non-adherent cell of suspension, add in attached cell AIM ?V substratum 4ml, this substratum include IL ?4 (20ng/ml) and GM ?CSF (100ng/ml).
After 3 days, attached cell supplement AIM ?V nutrient solution 2ml, this nutrient solution include IL ?4 (20ng/ml) and GM ?CSF (100ng/ml), be still placed in 37 DEG C, 50% (volumetric concentration) CO
2cultivate in incubator.
6th day: attached cell: add Hepatitis B virus vaccine stoste 10 μ g (the safe company in Shenzhen), cultivate 24 hours, obtain the functional DC of Antigen.
Fig. 1 shows DR, CD80 and CD86 containing ripe mark, and monocyte induced maturation is the functional DC with CD80 and CD86 mark.
Embodiment 2:T cell amplification
The suspension cell (i.e. T cell) obtained in embodiment 1 is moved to AIM containing 4ml ?VT75 culturing bottle, AIM ?after V substratum washs 3 times (amount of liquor 2ml/ time), add 1ug/mlOKT3,1ug/ml Kang ?CD28, be placed in 37 DEG C, 50% (volumetric concentration) CO
2cultivate in incubator.
2nd day: supplement in T cell nutrient solution AIM ?V nutrient solution 5 ?10ml, this nutrient solution include IL ?2 (300IU/ml) and 20% (mass body volume concentrations) albumin 1.5ml.
4th day: supplement AIM ?V nutrient solution 5 ~ 10ml, this nutrient solution include IL ?2 (300IU/ml) and 20% (mass body volume concentrations) albumin 1.5ml, continue at 37 DEG C, 50% (volumetric concentration) CO
2be cultured to the 8th day in incubator, obtain the T cell of propagation.
Embodiment 3:HBV specific T-cells
8th day: by " obtaining the functional DC of Antigen " of obtaining in embodiment 1 and the proliferative T cell mixing Dual culture obtained in embodiment 2.
10th day: supplement AIM ?V nutrient solution 5 ?10ml, this nutrient solution includes IL2 (300IU/ml) and 20% albumin 1.5ml.
12nd day: supplement AIM ?V nutrient solution 5 ?10ml, this nutrient solution includes IL2 (300IU/ml) and 20% albumin 1.5ml.
14th day: obtain HBV specific T-cells.
Fig. 2: HBV sensitization DC and T cell Dual culture can produce more CD8+ cytotoxic T cell.
As shown in Figure 2, the PBMC following methods of Patients with Chronic HBV Infection and existing hepatitis B protection antibody Healthy People is utilized to induce respectively: method 1: directly add Hepatitis B virus vaccine in PBMC; Method 2:PBMC adds Hepatitis B virus vaccine and IL-4; Method 3:HBV sensitization DC and T cell Dual culture.Result shows, and method 2 and 3 produces CD8+T cellular water on average higher than method 1, and 3 is more remarkable in method.
Fig. 3: the level identifying above different treatment group release IFN-γ.Result shows, no matter natural infection decubation Healthy People or chb patient, and employing method 2 and 3 induces the IFN-γ produced to be significantly higher than method 1, and the ELISPOT index that method 3 (HBV sensitization DC and T Dual culture) induces is the highest.
Embodiment 4: utilize the HBV specificity IFN ?γ level that the more alone medicine of cell in vitro system and medicine superposition HPCDT cell therapy produce.
PBMC (peripheral blood mononuclear cell) is separated with pharmacological agent superposition HPDCT cell therapy peripheral blood in patients respectively from alone pharmacological agent, after 20%PBMC stimulates 2 hours with HBV full-length genome peptide, again with the 80%PBMC Dual culture 10 days of remainder, nutrient solution is containing 2%AB serum, again with IFN ?γ streaming antibody incubation 6 ?8 hours, Flow Cytometry detect IFN ?γ level.As shown in Figure 4, antiviral superposition HPDCT shows stronger antiviral effect to detected result.
Embodiment 5: cell prepares the precaution of security detection and HPCDT cell adoptive therapy chb patient
Prepare the 11st day and the 14th day at cell, get cell culture fluid and carry out king crab experiment and Endotoxin test and microbial culture, guarantee the cell safety prepared.Before patient feeds back cell, first by the cell centrifugation in mixed culture, after brine 3 times, cell concentration is (1 ?2) * 10
7, be resuspended in the physiological saline of 10ml, add 20% albumin 1.5ml to prevent interionic sticking simultaneously, then add in 100ml physiological saline, in venous re-transfusion patient body with syringe.Precaution have: monitor vital sign (body temperature, breathing, pulse) (2) infusion process before, during and after (1) infusion and observe with or without following performance: dizzy, headache, uncomfortable in chest, palpitaition, Nausea and vomiting, stomachache, expiratory dyspnea, the disturbance of consciousness, shock and other symptoms.
Embodiment 6: the difference of antiviral superposition HPDCT cell therapy and alone drug effect sexual cell factor level
30 examples just control chb patient, and 15 examples are assigned randomly to alone antiviral (Entecavir, ETV), and other 15 examples superpose HPDCT cell therapy after giving ETV3 month, within every 2 weeks, give a cell therapy, totally 6 times; Observe 36 weeks (Fig. 5).ETV single therapy and ETV+HPDCT combination therapy flow process are as shown in Figure 5.
Within 36th week, compare two groups of patient T cells's effectors (IL ?17, TNF ?α and IFN ?γ) and T cell suppressive genes (IL ?10) level, prompting: ETV combine HPDCT treatment group patient produce degraded virus is played a crucial role T cell effector (IL ?17 and IFN ?γ) be significantly higher than ETV single therapy (Fig. 6).
As shown in Figure 6, ETV combine HPDCT treatment group patient produce degraded virus is played a crucial role T cell effector (IL ?17 and IFN ?γ) be significantly higher than ETV single therapy.
Claims (9)
1. a construction process for hepatitis vaccine primed dendritic shape cell induction specific T-cells, comprises the steps:
(1) antigen load monocyte derived dendritic cell is obtained;
(2) T cell amplification;
(3) C virusspecific T cell is obtained.
2. method according to claim 1, is characterized in that, described step (1) is: gather peripheral blood 10ml, separating peripheral blood mononuclear cells, be resuspended in 4ml AIM ?V nutrient solution, be then placed in 37 DEG C, 50%CO
2adherent culture 2 hours in incubator; After 2 hours take out, jog, removes suspension cell, attached cell, namely add in monocyte 4mlAIM ?V substratum, this substratum include concentration be 20ng/ml IL ?4 and concentration be 100ng/ml GM ?CSF; After 3 days, monocyte supplement AIM ?V nutrient solution 2ml, this nutrient solution containing concentration be 20ng/ml IL ?4 and concentration be 100ng/ml GM ?CSF, be still placed in 37 DEG C, 50%CO
2cultivate in incubator, making it ripe is monocyte inducing dendritic shape cell; In moDCs cell, add hepatitis virus vaccine stoste 10 μ g when the 6th day, cultivate 24 hours, namely obtain antigen load monocyte derived dendritic cell.
3. method according to claim 1, is characterized in that, described step (2) is: by the suspension cell obtained in step (1), i.e. T cell, move to containing 4ml AIM ?the T75 culturing bottle of V nutrient solution, with AIM ?V substratum wash 3 times, amount of liquor is 2ml/ time; Then add the anti-CD28 that OKT3 that final concentration is 1 μ g/ml and final concentration are 1 μ g/ml, be placed in 37 DEG C, 50%CO
2cultivate in incubator; 2nd day: T cell supplement AIM ?V nutrient solution 5 ?10ml, this nutrient solution containing concentration be 300IU/ml IL ?2 and mass body volume concentrations be the albumin 1.5ml of 20%; 4th day: T cell supplement AIM ?V nutrient solution 5 ?10ml, this nutrient solution include 300IU/ml IL ?2 and mass body volume concentrations be the albumin 1.5ml of 20%, continue at 37 DEG C, 50%CO
2be cultured to the 8th day in incubator, obtain the T cell of propagation.
4. method according to claim 1, it is characterized in that, described step (3) is: the T cell of the propagation obtained in the antigen load monocyte derived dendritic cell obtained in step (1) and step (2) is mixed Dual culture; 10th day: supplement AIM ?V nutrient solution 5 ?10ml, this nutrient solution containing concentration be 300IU/ml IL ?2 and mass body volume concentrations be the albumin 1.5ml of 20%; 12nd day: supplement AIM ?V nutrient solution 5 ?10ml, this nutrient solution containing concentration be 300IU/ml IL ?2 and mass body volume concentrations be 20% albumin 1.5ml; 14th day: obtain C virusspecific T cell.
5. the method according to any one of Claims 1-4, is characterized in that, described hepatitis vaccine is any one in hepatitis A vaccine, Hepatitis B virus vaccine or hepatitis C vaccine.
6. the cell in vitro system that the method according to any one of claim 1 to 5 is obtained.
7. comprise the cell preparation of cell in vitro system described in claim 6.
8. the purposes of the method described in any one of claim 1 to 5 in the cell preparation for the preparation for the treatment of hepatitis.
9. purposes according to claim 8, is characterized in that: described hepatitis is any one in hepatitis A, hepatitis B or the third liver.
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| CN104288179A (en) * | 2014-07-23 | 2015-01-21 | 武汉汉密顿生物科技股份有限公司 | Dendritic cell, preparation method thereof and application |
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| CN104288179A (en) * | 2014-07-23 | 2015-01-21 | 武汉汉密顿生物科技股份有限公司 | Dendritic cell, preparation method thereof and application |
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Application publication date: 20160302 |