CN103396992A - Culture and application of oligoclonal tumor-infiltrating lymphocytes for liver cancer - Google Patents
Culture and application of oligoclonal tumor-infiltrating lymphocytes for liver cancer Download PDFInfo
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Abstract
The invention relates to culture and an application of oligoclonal tumor-infiltrating lymphocytes for liver cancer, and provides an oligoclonal cultural method of tumor-infiltrating lymphocytes (TIL), wherein TIL cell population with strong antitumor activity is selectively amplified from liver cancer tissues, and is applied to adoptive immunotherapy of liver cancer.
Description
Technical field
The invention belongs to bioengineering field, be specifically related to a kind of can effectively the extraction with the lymphocytic method of amplification cultivation invasion in HCC and in the application aspect the liver cancer immunity treatment.
Background technology
Hepatocellular carcinoma (hepatocellular carcinoma, HCC) is one of five the most common in the world large malignant tumours, and China accounts for the whole world 45%.At present, the treatment of liver cancer oneself to develop into to perform the operation from single excision be the main multimodal complex therapy stages [1] such as hepatic arterial chemoembolization, outer radiotherapy, topical therapeutic and immunotherapy that comprise.These means in effective killing tumor cells, are inevitably brought in various degree damage to body's immunity.Along with immunology, molecular biological develop rapidly, after operation, radiotherapy, the large therapy of chemotherapy three, oneself becomes the 4th kind of pattern of oncotherapy the biotherapy of tumour.The biotherapy of tumour comprise immunotherapy, gene therapy, stem-cell therapy, inducing tumor cell differentiation and apoptosis, suppress tumor neogenetic blood vessels treatment etc., be mainly wherein immunotherapy [2].The hepatocarcinoma patient immunologic hypofunction, particularly the local immunity microenvironment is abnormal, and causing the immunity of organism defensive raction can not be effectively the important factor [3] that liver cancer produces immunologic escape, easy transfer and relapse.How effectively regulating host immune function, improve the performance of liver local immunity microenvironment in order to the anti-knurl effect of body, is important, the effective means of preventing and treating the liver cancer recurrence and metastasis after resection, eliminating postoperative residual knurl, is the important content of liver cancer complex therapy.
, although liver cancer immunity research has obtained certain progress, to liver cancer cell actually, be how the definite mechanism that monitors of inducing immune tolerance and escape from immune is not yet fully understood.When inducing the special t cell responses of activation antigen, must pay attention to the restraining effect of liver local immunity microenvironment to the T cell of activation.Only in this way, could effectively excite antineoplastic immune, produce anti-knurl effect preferably.Therefore use immune means, the immunologic function that improves body itself becomes the new measure for the treatment of tumour, makes the immune protection research increasingly important [4] of HCC.Tumor infiltrating lymphocyte (tumor infiltrating lymphocyte, TIL) is that a group is infiltrated in tumor tissues, and autologous tumor cell is had than the lymphocyte of high specific killing activity.Because itself and tumour cell close contact are considered to the direct and special performance of host to tumour identification, have the advantages such as efficient, special, that side effect is little, be subject in recent years Chinese scholars special concern [5].In tumor tissues, the variation of lymphocyte infiltration quantity and function has reflected intensity and the aggregate level of the antitumor reaction of body to a certain extent.Yet freshly extd TIL anti-tumor activity is very low, activate TIL and carry out adoptive immunotherapy, but clinical therapeutic efficacy is unsatisfactory, studies show that the immunity system of malignant tumor patient all is in tolerance or defect state [6] at tumor by local and whole body.Induce TIL that immune bias occurs bringing out stage regulatory T cells (regulatory T cells, Treg), what have makes several nonfunctionals of TIL because Treg quantity significantly increases, and makes the anti-knurl immunity of TIL get into a difficult position [7].
Take cellular immunization in main tumour immunity process, Treg is in key link.Sakaguchi in 1999 etc. will remove CD25
+The single positive T cell adoptive transfer of the CD4 of cell is to nude mice, and the various autoimmune of nude mice generation as a result is sick, and with CD4
+CD25
+T cell and CD4
+Autoimmune disease, just do not occur in the common adoptive transfer of single positive T cell, confirms CD4
+CD25
+The T cell can be kept autoimmune tolerance.CD4 is also found in vitro study subsequently
+CD25
+The T cell has low reactivity and immune suppression function, according to its function with CD4
+CD25
+The T cell is as regulatory T cells [8].Its infiltration degree in tumor tissues has represented aspect of body tumour immunity inhibited reaction, plays an important role in Organism immunoregulation, determines that immunoreactive final trend is activate immunity or induces tolerance.The discoveries such as Jone are removed the Treg cell with Anti-CD25 monoclonal antibody can suppress the growth of malignant melanoma; Treatment provides new immunotherapy [9] to human tumor for this.The Treg cell is united the immunogenic method of other raising tumour cells can improve tumour immunity.There is research group also testing a kind of retarding agent of Treg cell, can treat malignant melanoma [10].Status and effect in view of the Treg cell in immunology, its research and application have become the popular direction of nearest tumour immunity and immunotherapy, and have obtained gratifying achievement.
The TIL Activation In Vitro is cultivated the propagation of TIL, killing ability is recovered, illustrate TIL be subjected to or under attack be that local environment causes, the function of TIL, phenotype variation and cell bias also can be reinvented fully.Yet the method that Activation In Vitro is cultivated does not change TIL suppressed environment in vivo.TIL separation method commonly used comprises digestion tumor tissues such as utilizing collagenase, lymphocyte separation medium and density gradient centrifugation separation TIL at present, then at the substratum (the RPMI1640 nutrient solution that contains 10% human serum) of external use synthetic, then two all left and right times of cultivation recover in patient body to carry out immunotherapy [11].Specific as follows: get fresh tumor tissues sample, remove normal surrounding tissue and necrotic tissue, the PBS washing, rinse well with the RPMI1640 nutrient solution, moves in sterile vessel and with operating scissors, tumor tissues is shredded to 1-2mm
3Fritter, be placed in the RPMI1640 nutrient solution 40ml of digestive ferment, stirring and evenly mixing at room temperature, and 4 ℃ are spent the night.Remove indigested tissue block, the application lymphocyte separation medium makes 2 * 10
6The cell suspension of/ml left and right, after gradient density centrifugal, draw 75% and 100% and separate between liquid level the white cellular layer that is rich in TIL,, with HankShi liquid washing, centrifugal, cultivates afterwards amplification.Carry out in addition improved " cultivating the method for til cell from body hydrothorax supernatant " (patent of invention number: CN200410014095.9), use the supernatant of the centrifugal rear preservation of cancerous hydrothorax of collection instead as nutrient solution, the vitro culture til cell on this method basis.And another cultural method " a kind of method of effective cultivation tumor infiltrating lymphocyte " (patent application number: CN201110028257.4) add recombinant human scleroproein and cluster differentiation group 3 antibody in the nutrient solution of a upper patent of invention, carry out amplification in vitro and the cultivation of til cell, added again the apoptosis regulatory protein survivin in 1-2 days in nutrient solution and read albumen-1 before collecting til cell.No matter be that tradition or the TIL of the cultivation amplification of advancing improvement feed back human body, its antitumor curative effect is barely satisfactory, and its basic reason is as follows: the digestion tumor tissues such as (1) collagenase have damage to TIL; (2) separate the TIL purity obtain not high; (3), to the amplification non-selectivity of TIL, can not increase for having high antitumor activity TIL.
Can obtain a large amount of cells although prepare at present the method for the TIL that increases, but still have defect, such as can not the selective amplification anti-tumor activity strong TIL colony etc.Therefore, the TIL that how to obtain to have in a large number high anti-tumor activity is the adopt key for the treatment of of immunity.
Summary of the invention
The object of the present invention is to provide cultivation and the application of few clone's liver cancer tumor infiltrating lymphocyte.
In a first aspect of the present invention, a kind of cultural method of liver cancer tumor infiltrating lymphocyte is provided, described method comprises:
(1) from liver cancer in vitro tissue sample, obtain the tissue of the inside and outside 2mm scope in tumour and position, healthy tissues boundary;
(2) tissue of culturing step (1), add IL-2 in substratum, promotes lymphocyte growth, ooze out and breed, and obtains the liver cancer tumor infiltrating lymphocyte.
In a preference, in step (2), the final concentration of IL-2 in substratum is 6000 ± 1000IU/ml.
In another preference, in step (2), described substratum be RPMI 1640 substratum (preferably, pH7.2), wherein also contain: 25 ± 5mmol/L HEPES, 100 ± 20U/ml penicillin, 100 ± 20U/ml Streptomycin sulphate, 2 ± 0.4mmol/L glutamine, (5.5 ± 1) * 10
-5Mol/L beta-mercaptoethanol, 10 ± 2% people AB serum.
In another preference, in step (2), the tissue of step (1) is placed in 37 ℃, the 5%CO of 24 orifice plates in humidification
2Cultivate in environment.
In another preference, in culturing process, change weekly half substratum.
In another preference, in the described culture that contains the liver cancer tumor infiltrating lymphocyte, Treg cell proportion is lower than 10%.
In another preference, in the described culture that contains the liver cancer tumor infiltrating lymphocyte, IFN-γ concentration is higher than 280pg/mL.
In another preference, described method does not adopt enzyme (as collagenase) to carry out the digestion of tissue samples.
The culture that contains the liver cancer tumor infiltrating lymphocyte of described method preparation is provided in another aspect of this invention.
In another aspect of this invention, provide the described purposes that contains the culture of liver cancer tumor infiltrating lymphocyte, for the preparation of the adopt medicine of Hepatoma therapy of immunity.
In another preference, a kind of test kit of the culture for the preparation of containing the liver cancer tumor infiltrating lymphocyte, described test kit comprises:
IL-2; (preferably, pH7.2), wherein also contain: 25 ± 5mmol/L HEPES, 100 ± 20U/ml penicillin, 100 ± 20U/ml Streptomycin sulphate, 2 ± 0.4mmol/L glutamine, (5.5 ± 1) * 10 with RPMI 1640 substratum
-5Mol/L beta-mercaptoethanol, 10 ± 2% people AB serum.
Other side of the present invention, due to the disclosure of this paper, is apparent to those skilled in the art.
Description of drawings
Fig. 1, liver cancer patient tumor epithelial cell (tumor center's tissue) and tumour perienchyma (tumour and position, healthy tissues boundary tissue) Treg cell absolute number analysis.The inner Treg density of tumor epithelial cell is higher than tumour perienchyma (P=0.009).
Fig. 2, with the tissue block of 2mm scope inside and outside tumour and healthy tissues boundary line, cultivate the morphologic observations of the liver cancer tumor infiltrating lymphocyte of different few clone's incubation times.A: the 1st day; B: the 3rd day; C: the 7th day; D: the 14th day.
Fig. 3, with the tissue block of 2mm scope inside and outside tumour and healthy tissues boundary line, cultivate, few clone cultivates and the TIL of traditional cultural method carries out the flow cytometer showed evaluation.A: few clone's culture method blank group; B: few clone's culture method group; C: general culture method blank group; D: general culture method group.
Fig. 4, tumour cell be as target cell, and tumor infiltrating lymphocyte action effect cell, few cloning process are cultivated and ordinary method is cultivated, the liver cancer cell comparison of situation of surviving after negative blank.
Between Fig. 5, different few clone's liver cancer TIL, the target cell toxic action is different.
The Treg ratio of Fig. 6, few clone TIL cultured continuously changes.The point of right upper quadrant represents CD4
+CD25
+Foxp3
+The Treg cell.As seen in the TIL in 2 weeks, 3 weeks, 4 weeks, 5 weeks, the Treg ratio descends gradually.A:2 week (Treg cell proportion 5.55%); B:3 week (Treg cell proportion 1.81%); C:4 week (Treg cell proportion 0.44%); D:5 week (Treg cell proportion 0.23%).
Fig. 7, ELISA test kit detection by quantitative tumor infiltrating lymphocyte anti-tumor activity, namely measure the ratio of contained Treg in different few clone's til cells and with liver cancer cell, cultivate altogether the IFN-γ concentration that discharges.In figure, the unit of Treg ratio is " % "; IFN γ concentration unit is pg/mL.
Between Fig. 8, different few clone's liver cancer TIL, IFN-γ secretory volume is different.
Fig. 9, the ability of tumor infiltrating lymphocyte secretion of gamma-IFN that contains higher proportion Treg cell are lower, and anti-tumor activity is poor.Each point in figure represents that different few clones cultivate the ratio of Treg in TIL and the concentration that TIL discharges IFN-γ.During few clone TIL cultivates, Treg accounts for percentage of lymphocyte and becomes negative correlation with TIL release IFN-γ concentration, and relation conefficient is-0.650, P value＜0.001.
Figure 10, the immunity of different TIL treatment group are adopted and are treated the gross tumor volume (TIL of H22 liver cancer tumor-bearing mice front and back
△: few clone TIL treatment group; TIL: conventional TIL treatment group; The white control group of Control:PBS liquid air).
Figure 11, different TIL treatment group mouse tumor volume change (TIL with treatment time
△: few clone TIL treatment group; TIL: conventional TIL treatment group; The white control group of Control:PBS liquid air).
The tumour inhibiting rate of Figure 12, different TIL treatment groups (the white control group of Control:PBS liquid air; TIL: conventional TIL treatment group; TIL
△: few clone TIL treatment group).
Figure 13, immunohistochemical methods detect the expression of FoxP3 in different TIL treatment group mouse tumors.The white control group FoxP3 of A:PBS liquid air (+++) express, shown in arrow; B: conventional TIL treatment group FoxP3 (+) expresses; C: few clone TIL treatment group FoxP3 (+) expresses.Left: * 200; Right: * 400.
Figure 14, different TIL treatment group mouse peripheral blood cytokine change (the white control group of Control:PBS liquid air; TIL: conventional TIL treatment group; TIL
△: few clone TIL treatment group).
Embodiment
Defect for existing cultural method existence, the widow that the present invention has disclosed a kind of tumor infiltrating lymphocyte (TIL) clones cultural method, the til cell group that the selective amplification anti-tumor activity is strong from liver cancer tissue, and be applied to the adoptive immunotherapy of liver cancer.
Adoptive immunotherapy needs a reliable and stable TIL cultural method.Few clone TIL culture method has the characteristics of himself, from the independent few clone of same tumour different sites, cultivates and usually can show different proliferative responses and antigen-specific anti-tumor activity.The widow that the unhomogeneity of local T cell may cause originating from same tumour different sites clones the difference of TIL anti-tumor activity.
So-called few clone's tumor infiltrating lymphocyte is a group TIL that a group cultivates out with minority TIL clonal expansion.The inventor is by at Initial stage of culture, and by at the tumour different sites, drawing materials, and the volume of controlling the cultured tissue piece limits the Treg number of cultivating when initial.Pointed out the position of drawing materials of carrying out few clone TIL cultivation should select the juncture area of tumour and nonneoplastic tissue.
Few clone TIL culture method of the present invention, carry out a plurality of from same tumour and few clone TIL independent of each other cultivation simultaneously by (1); (2) detect the anti-tumor activity of each few clone TIL; (3) the further high TIL of amplification cultivation anti-tumor activity, determined can obtain the tissue of 2mm scope inside and outside application tumour and position, healthy tissues boundary the good widow of anti-tumor activity and clone TIL.
Therefore with respect to existing cultural method, the present invention has following advantage: (1) need not be by digestion tumor tissues such as collagenases, allow TIL voluntarily migration go out tumor tissues, and further propagation, separate the process of TIL to the TIL not damaged; (2) because til cell is suspension growth,, by controlling nutrient solution condition and the cultivation of repeatedly going down to posterity, can reject other impurity cells, obtain the til cell of higher degree; (3) cultivate altogether by autologous tumor cell and TIL, and detect TIL secretion of gamma-IFN situation, can differentiate the anti-tumor activity of each different few clone TIL, and then optionally the widow of the high antitumor activity of amplification cultivation clones TIL.The til cell anti-tumor activity of (4) turning out is high, has strengthened the immunity treatment function of adopting.
Apply method of the present invention, can obtain to contain the TIL of lower Treg ratio.Experimentation on animals shows, the anti-tumor activity that the til cell of the present invention's preparation has excellence.
The present invention also provides and has applied the culture that contains the liver cancer tumor infiltrating lymphocyte that method of the present invention prepares.In described culture, Treg cell proportion is lower than 10%; And IFN-γ concentration is higher than 280pg/mL.
Based on the inventor's new discovery, the present invention also provides the test kit for the preparation of the culture that contains the liver cancer tumor infiltrating lymphocyte, and described test kit comprises: IL-2; , with RPMI 1640 substratum, wherein also contain: 25 ± 5mmol/L HEPES, 100 ± 20U/ml penicillin, 100 ± 20 μ lg/ml Streptomycin sulphates, 2 ± 0.4mmol/L glutamine, (5.5 ± 1) * 10
-5Mol/L beta-mercaptoethanol, 10 ± 2% people AB serum.
After the tissue that has obtained 2mm scope inside and outside the tumour that exsomatizes and position, healthy tissues boundary, the reagent in this organizations test kit of the present invention is carried out the cultivation of TIL.Preferably, the working instructions that also can comprise explanation TIL cultural method in described test kit, cultivate to instruct those skilled in the art to carry out other noumenal tumours (as tumours such as cancer of the stomach, intestinal cancer, carcinoma of the pancreas, melanoma, mammary cancer) TIL with method of the present invention, obtain to have the TIL of excellent anti-tumor activity.
, below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only are not used in and limit the scope of the invention for explanation the present invention.The experimental technique of unreceipted actual conditions in the following example, write according to normal condition such as J. Pehanorm Brooker etc. usually, molecular cloning experiment guide, the third edition, Science Press, the condition described in 2002, or the condition of advising according to manufacturer.
Relation and the distribution characteristics of Treg in the liver cancer tumor tissues of embodiment 1, the lymphocytic cultivation of few clone's invasion in HCC, TIL dysfunction and Treg
One, few clone's culture method
Few clone's culture method comprises the following steps:
1, get fresh HCC tumor tissues specimens from pri, remove normal surrounding tissue and necrotic tissue, PBS washing 3 times.
2, get the tissue block of diameter 2mm at the tumour different sites, scope of selecting material comprises tumor center's tissue and tumour juncture area (the inside and outside 2mm scope of tumour and healthy tissues boundary line).
3, add the 2ml perfect medium in 24 orifice plates, wherein contain IL-2 (6000IU/ml).Perfect medium (pH7.2) is RPMI 1640 substratum, also comprises: 25mmol/L HEPES (4-hydroxyethyl piperazine ethanesulfonic acid), pH7.2,100U/ml penicillin, 100U/ml Streptomycin sulphate, 2mmol/L glutamine, 5.5 * 10
-5Mol/L beta-mercaptoethanol, 10% people AB serum form.Put into 1 tumor tissues fritter in every hole, afterwards 24 orifice plates are placed in 37 ℃, 5%CO of humidification
2In incubator, until lymphocyte is obviously grown.
4, observe under inverted microscope every 1 day that each lump is lymphocytic to ooze out and breed.Visible lymphocyte growth no matter whether, 1 all domestic demands are changed half nutrient solution.Cultivating 1-2 after week, intensive lymphocyte covers on each tissue block a part of flat board on every side.
5, when the cell in hole almost converges,, with its mixing, to go down to posterity and be assigned to 2 holes, every hole adds the 2ml perfect medium that contains IL-2 (6000IU/ml).
6,, according to Growth of Cells situation separation and Culture thing, make cell density maintain 0.8～1.6 * 10
6Individual/hole, half nutrient solution of 2 times/week displacement.Each archioporus, as an independently TIL cultivation, keeps separating with other hole.Be the cell of suspension growth because of til cell, during subsequent experimental, get the substratum suspension that is suspended with til cell and get final product.
Induce TIL that immune bias occurs bringing out stage regulatory T cells (Treg), what have makes several nonfunctionals of TIL because Treg quantity significantly increases, and the anti-knurl immunity of TIL is got into a difficult position.By above-mentioned test, the inventor is definite, and in tumor microenvironment of hepatocellular carcinoma, Treg distributes and has characteristic, be that in tumor epithelial cell, Treg density is higher than the other regional organization of cancer, Treg is distributed in mesenchyma stroma of tumors in a large number in liver cancer tissue, and it is rare to distribute in cancer beside organism, and be scattered distribution, as Fig. 1.
Therefore, it is better that Initial stage of culture contains TIL proliferative and the anti-tumor activity of lower Treg ratio.
General culture method is cultivated TIL and comprised the following steps: get fresh HCC tumor tissues sample, remove normal surrounding tissue and necrotic tissue, the PBS washing, rinse well with the RPMI1640 nutrient solution, moves in sterile vessel and with operating scissors, tumor tissues is shredded to 1-2mm
3Fritter, be placed in the RPMI1640 nutrient solution 40ml of digestive ferment, stirring and evenly mixing at room temperature, and 4 ℃ are spent the night.Remove indigested tissue block, the application lymphocyte separation medium makes 2 * 10
6The cell suspension of/ml left and right, after gradient density centrifugal, draw 75% and 100% and separate between liquid level the white cellular layer that is rich in TIL,, with HankShi liquid washing, centrifugal, cultivates afterwards amplification.
The separation of embodiment 3, tumor infiltrating lymphocyte, culture ﹠ identification
Get the inside and outside 2mm scope of liver cancer tumour and healthy tissues boundary line, as embodiment 1 method, cultivate TIL, dynamically observe.In the TIL culturing process, TIL migration gradually goes out tumor tissue, and forms an intensive lymphocyte populations around tumor tissue.As seen from Figure 2, cultivated the 1st day, visible several lymphocyte migration go out tumor tissues around tumor mass; Cultivated 3 days, and as seen than the multi-lymphocytes migration, went out tumor tissues, be distributed in around tumor tissues; Cultivated the 7th day, visible lymphocyte density further increases.Cultivated the 14th day, lymphocyte quantity further increases, and almost converges.Along with the prolongation of incubation time, the lymphocyte quantity in culture plate is on the increase.Clone the prepared tumor infiltrating lymphocyte cell density of cultural method by the widow and maintain 0.8～1.6 * 10
6Individual/hole, and carry out amplification in vitro and cultivate.
Flow cytometer detects to be found, the height (Fig. 3) that tumor infiltrating lymphocyte (tissue block with 2mm scope inside and outside tumour and the healthy tissues boundary line is cultivated) purity that few clone's cultural method is turned out is cultivated than ordinary method, and have stronger anti-tumor activity (Fig. 4).
The anti-tumor activity of embodiment 4, the few clone of detection TIL
Tissue block with 2mm scope inside and outside liver cancer tumour and healthy tissues boundary line is cultivated, and the widow who chose at random for 30 example 2 weeks of cultivation clones liver cancer TIL, detects the cytotoxicity to autogenous hepatocarcinoma cell.Result shows, few clone's liver cancer TIL has significant lethal effect to autogenous hepatocarcinoma cell, and the lethal effects not identical (Fig. 5) between different few clone's liver cancer TIL under same incubation time, and statistical significance (P＜0.01) is arranged.
Choose at random in 6 patients' liver cancer tissue (liver cancer tumour and healthy tissues boundary line inside and outside 2mm scope) and respectively get 2 few clones and cultivate samples (totally 12 few clones of TIL) and carry out sequential culture and observe, detect Treg proportion in its TIL when cultivating 2w, 3w, 4w, 5w.In the TIL of 2w and 3w, there is notable difference (P＜0.001) in the Treg proportion, in the TIL of 3w and 4w, there is notable difference (P=0.001) in the Treg proportion, in the TIL of 4w and 5w, the difference of Treg proportion does not have statistical significance (P=0.148), sees Fig. 6.Result shows, in vitro culture TIL process, in the same few clone TIL of different incubation times, the Treg proportion is not identical, and the Treg proportion descends with the prolongation of incubation time.
The widow who originates from same liver cancer tissue different sites clones and cultivates in TIL, and the Treg proportion is different; The til cell that same few clone cultivates, the Treg proportion descends gradually with the prolongation of incubation time.The widow who derives from same liver cancer tissue different sites clones the ability difference of cultivating the TIL secretion of gamma-IFN.IFN-γ is mainly produced by T cell and NK cell, is one of main immunomodifier of secretion after TIL activates, has immune synergism.On the other hand, the variation of the appearance of IFN-γ and secretory volume is also indicating the state of activation of TIL.Therefore, detect its secretory volume and changing conditions and can assist well to judge the state of activation of TIL.
In the present embodiment, detect simultaneously 30 examples and cultivate the secretory volume that the widow in 2 weeks clones the Treg cell proportion in liver cancer TIL and with autogenous hepatocarcinoma cell, cultivates altogether rear IFN-γ, observe different few clone's liver cancer TIL secretion of gamma-IFN not identical (Fig. 7-8) under same incubation time, statistical significance (P＜0.01) is arranged.
In the same few clone TIL of different incubation times, Treg cell proportion is not identical yet.Get different few clones and cultivate TIL and carry out Treg ratio measuring (flow cytometer), and the IFN-γ release conditions after measuring same few clone and cultivating TIL and liver cancer cell and cultivate altogether.To the Treg ratio with result after IFN-γ concentration is carried out correlation analysis, be: during few clone TIL cultivates, the Treg cell accounts for percentage of lymphocyte (X-coordinate) and becomes negative correlation with tumor infiltrating lymphocyte release IFN-γ concentration (ordinate zou), sees Fig. 9.Relation conefficient is-0.650, P＜0.001, and statistical significance is arranged.As shown in Figure 9, the ability of tumor infiltrating lymphocyte secretion of gamma-IFN that contains higher proportion Treg cell is lower, and anti-tumor activity is poor.
Choose at random approximately 18 of the H22 liver cancer tumor-bearing mices of 1.0cm (The 2nd Army Medical College experimentation on animals center) of tumor average diameter, in mouse 6 weeks of age, diameter of tumor is 1.0cm approximately, is divided at random 3 groups, 6 every group.
Be respectively control group, conventional TIL treatment group (with the tumor material obtaining of this H22 tumor-bearing mice, with embodiment 2 methods preparations), few clone (tissue block with 2mm scope inside and outside the tumour of this H22 tumor-bearing mice and healthy tissues boundary line is drawn materials, with embodiment 1 method, cultivated preparation) treatment group, give respectively the TIL of PBS liquid, cellar culture and each 0.3mL of TIL that few clone cultivates by tail vein injection, til cell quantity is 1 * 10
7, every 7d 1 time, totally 3 times.Measure tumour major diameter (a) and minor axis (b) every 3d after injection, according to formula gross tumor volume V=0.5 * a * b
2Calculate gross tumor volume, draw tumor growth curve; Measure simultaneously the body weight of each mouse, observe its body weight change.Put to death mouse after 30d, pluck knurl and claim the knurl weight, calculate the inhibiting rate of different TIL treatment groups to tumour.Tumour inhibiting rate (%)=(control group knurl weight-average value-experimental group knurl weight-average value)/control group knurl weight-average value * 100%.
1.TIL the restraining effect to murine transplanted hepatocarcinoma
Tumor-bearing mice is after giving TIL treatment 30d, and the growth that can be observed tumour obviously is suppressed, and conventional TIL treatment group is respectively (1.172 ± 0.085) cm with the gross tumor volume (Figure 10-11) of few clone TIL treatment group
3(0.891 ± 0.061) cm
3, difference has statistical significance (P＜0.05); Knurl heavily is respectively (1.31 ± 0.22) g and (0.91 ± 0.09) g, and difference has statistical significance (P＜0.05); All lower than control group gross tumor volume (1.714 ± 0.040) cm
3With heavy (2.43 ± 0.17) g of knurl, difference all has statistical significance (P＜0.01).
Few clone's TIL treatment group tumour inhibiting rate (62.52 ± 3.76) % is apparently higher than conventional TIL treatment group (46.13 ± 9.21) %, and difference has statistical significance (P＜0.05), as Figure 12.
2. immunohistochemical methods detects the expression of FoxP3 in different treatment group H22 mouse tumors
Treg has the function that mediation tumour immunity is escaped, and FoxP3 is the specific marker thing of Treg.Therefore, by detecting the expression of FoxP3, can reflect TIL anti-tumor in vivo immunocompetence.The chemical detected result of immune group shows, 30d after the TIL treatment, during in the mouse tumor tissue, the FoxP3 positive rate reaches～and the strong positive rate is all lower than control group (Figure 13), and difference all has statistical significance (P＜0.01); In conventional TIL treatment group tumor tissues the FoxP3 positive rate and in～the strong positive rate all clones TIL treatment group (Figure 13) higher than the widow, difference has statistical significance (P＜0.05).
3. after the treatment, the serum cytokines expression level changes
Cytokines content detection result (Figure 14) shows, 30d after treatment, conventional TIL treatment group and few clone TIL treatment group peripheral blood TGF-β content are respectively (241.44 ± 56.01) pg/mL and (65.73 ± 44.79) pg/mL, and difference has statistical significance (P＜0.01); IL-10 content is respectively (166.52 ± 59.20) pg/mL and (36.66 ± 18.63) pg/mL, and difference has statistical significance (P＜0.01); All lower than control group (388.21 ± 18.67) pg/mL and (290.74 ± 65.90) pg/mL, difference all has statistical significance (P＜0.05).Conventional TIL treatment group and few clone TIL treatment group peripheral blood IFN-γ content are respectively (538.57 ± 103.43) pg/mL and (876.32 ± 114.52) pg/mL, and difference has statistical significance (P＜0.01); All higher than control group (106.66 ± 12.93) pg/mL, difference all has statistical significance (P＜0.05).Show by TIL and treat, can effectively improve the IFN-γ of Th1 emiocytosis in the mouse immune reaction, and suppress Th2 emiocytosis IL-10, TGF-β.
All quote in this application as a reference at all documents that the present invention mentions, just as each piece document is quoted separately as a reference.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
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Claims (10)
1. the cultural method of a liver cancer tumor infiltrating lymphocyte, is characterized in that, described method comprises:
(1) from liver cancer in vitro tissue sample, obtain the tissue of the inside and outside 2mm scope in tumour and position, healthy tissues boundary;
(2) tissue of culturing step (1), add IL-2 in substratum, promotes lymphocyte growth, ooze out and breed, and obtains the liver cancer tumor infiltrating lymphocyte.
2. the method for claim 1, is characterized in that, in step (2), the final concentration of IL-2 in substratum is 6000 ± 1000IU/ml.
3. the method for claim 1, it is characterized in that, in step (2), described substratum is RPMI 1640 substratum, wherein also contains: 25 ± 5mmol/L HEPES, 100 ± 20U/ml penicillin, 100 ± 20U/ml Streptomycin sulphate, 2 ± 0.4mmol/L glutamine, (5.5 ± 1) * 10
-5Mol/L beta-mercaptoethanol, 10 ± 2% people AB serum.
4. the method for claim 1, is characterized in that, in step (2), the tissue of step (1) is placed in 37 ℃, the 5%CO of 24 orifice plates in humidification
2Cultivate in environment.
5. the method for claim 1, is characterized in that, in culturing process, changes weekly half substratum.
6. the method for claim 1, is characterized in that, in the described culture that contains the liver cancer tumor infiltrating lymphocyte, Treg cell proportion is lower than 10%.
7. the method for claim 1, is characterized in that, in the described culture that contains the liver cancer tumor infiltrating lymphocyte, IFN-γ concentration is higher than 280pg/mL.
8. the culture that contains the liver cancer tumor infiltrating lymphocyte of the arbitrary described method preparation of claim 1-7.
9. the purposes that contains the culture of liver cancer tumor infiltrating lymphocyte claimed in claim 8, for the preparation of the adopt medicine of Hepatoma therapy of immunity.
10. the test kit for the preparation of the culture that contains the liver cancer tumor infiltrating lymphocyte, is characterized in that, described test kit comprises:
IL-2; , with RPMI 1640 substratum, wherein also contain: 25 ± 5mmol/L HEPES, 100 ± 20U/ml penicillin, 100 ± 20U/ml Streptomycin sulphate, 2 ± 0.4mmol/L glutamine, (5.5 ± 1) * 10
-5Mol/L beta-mercaptoethanol, 10 ± 2% people AB serum.
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Cited By (6)
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| CN103823068A (en) * | 2013-12-16 | 2014-05-28 | 周菊华 | Method for identifying anti-tumor lymphocytes |
| CN104278012A (en) * | 2014-10-11 | 2015-01-14 | 湖南赛诺生物科技有限责任公司 | Adult regulatory T cell in-vitro amplification culture medium and application method thereof |
| CN107502589A (en) * | 2017-08-04 | 2017-12-22 | 北京世纪劲得生物技术有限公司 | A kind of tumor infiltrating lymphocyte and mononuclearcell co-culture method |
| CN109609451A (en) * | 2019-01-24 | 2019-04-12 | 清华大学 | The method of the tumor infiltrating lymphocyte of external rapid amplifying kidney |
| CN112080467A (en) * | 2014-03-20 | 2020-12-15 | H.李莫菲特癌症中心研究院 | Tumor infiltrating lymphocytes for adoptive cell therapy |
| CN113005085A (en) * | 2021-03-18 | 2021-06-22 | 南方医科大学南方医院 | Novel method for culturing and in-vitro amplifying primary liver cancer tumor infiltrating lymphocytes |
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| CN103823068A (en) * | 2013-12-16 | 2014-05-28 | 周菊华 | Method for identifying anti-tumor lymphocytes |
| CN103823068B (en) * | 2013-12-16 | 2016-05-25 | 周菊华 | A kind of antitumor lymphocytic authentication method |
| CN112080467A (en) * | 2014-03-20 | 2020-12-15 | H.李莫菲特癌症中心研究院 | Tumor infiltrating lymphocytes for adoptive cell therapy |
| CN104278012A (en) * | 2014-10-11 | 2015-01-14 | 湖南赛诺生物科技有限责任公司 | Adult regulatory T cell in-vitro amplification culture medium and application method thereof |
| CN104278012B (en) * | 2014-10-11 | 2016-11-23 | 湖南赛诺生物科技股份有限公司 | A kind of adult's regulatory T cells amplification in vitro culture medium and using method thereof |
| CN107502589A (en) * | 2017-08-04 | 2017-12-22 | 北京世纪劲得生物技术有限公司 | A kind of tumor infiltrating lymphocyte and mononuclearcell co-culture method |
| CN109609451A (en) * | 2019-01-24 | 2019-04-12 | 清华大学 | The method of the tumor infiltrating lymphocyte of external rapid amplifying kidney |
| CN113005085A (en) * | 2021-03-18 | 2021-06-22 | 南方医科大学南方医院 | Novel method for culturing and in-vitro amplifying primary liver cancer tumor infiltrating lymphocytes |
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