CN103828763A - Liver cancer patient source heterotransplantation tumour mouse model and construction method thereof - Google Patents
Liver cancer patient source heterotransplantation tumour mouse model and construction method thereof Download PDFInfo
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Abstract
The invention provides a liver cancer patient source heterotransplantation tumour mouse model and a construction method of the mouse model. The liver cancer patient source heterotransplantation tumour mouse model comprises a mouse, wherein treated liver tissue blocks are vaccinated under the skin of the fore limb, the shoulders and the back of the mouse. The construction method comprises the steps of taking fresh liver cancer tissue, processing the tissue, vaccinating the treated liver cancer tissue blocks under the skin below the fore limb, the shoulders and the back of the mouse without the tumor, conducting conventional breeding, and obtaining the liver cancer patient source heterotransplantation tumour mouse model. The construction process is simple and easy, the tumor forming occurrence rate reaches 40 percent to 50 percent, and the method can be used for liver cancer medicine screening and liver cancer mechanism research. The built liver cancer patient source heterotransplantation tumour mouse model has the advantages of being simple and easy to manufacture, low in death rate caused by operation, high in success rate, easy to popularize, capable of being manufactured in mass, and good in synchronism, and is suitable for medicine screening and experiment research.
Description
Technical field
The present invention relates to a kind of liver cancer patient source xenograft tumor mouse model and construction method thereof.
Background technology
Primary carcinoma of liver is the malignant tumour of ranking the whole world the 5th, China the 3rd.Estimate according to the World Health Organization, before the year two thousand thirty, liver cancer all will keep sustainable growth, and its case fatality rate is in second especially.At present, operation is still one of primary treatment method of liver cancer, but exceed 60% liver cancer patient first entered middle and advanced stage when medical and with shifting, recurrence, thereby lose the chance of operative treatment.
Animal model for tumour is the Important Platform that carries out Tumor-assaciated mechanism and drug screening research.At present, clinical pre-neoplastic animal model adopts the method for tumor cell line suspension modeling more, and it has that tumor cell line easily obtains and experiment such as easily repeats at the advantage.But there is many defects in the tumor model that utilizes Establishment of Cell Line.Tumour cell is built in vitro the process that is and has been departed from natural tumor microenvironment, causes its gene to change; In Process of in vitro, selection pressure and the processing of process digestive ferment, destroy tumor tissues and cell heterogeneity and original structure, and the animal model that a strain cell-line is set up only can reflect a cancer patient; The tumour that cell-line becomes lacks the relevant microenvironment component of primary tumor.Finally cause oncobiology behavior and natural quality irreversibility to change, seriously hinder the mankind to tumour mechanism and treat the research of replying, it has become affects the bottleneck that therapeutic efficacy for hepatic carcinoma improves, urgently to be resolved hurrily.
Liver cancer patient source xenograft tumor mouse model is directly planted in excision liver cancer tissue in immunodeficient mouse body, at utmost simulation human internal environment, the specific gene, molecule and the histology heterogeneity thereof that retain tumour, can be widely used in etiology of liver cancer, pathogenesis, evolution and treatment.Compared with the animal model for tumour of setting up with traditional human carcinoma cell line, humanized's animal model for tumour is owing to having reduced extracorporeal treatment step, can keep better protistology feature and the histologic characteristics of clinical tumor tissue, farthest preserve the microenvironment of tumour, comprised lymphocyte, fibroblast, extracellular matrix and the vascular system of tumour cell, infiltration; And humanized's transplantable tumor model comes from different patients and does not cultivate through artificial, can reflect patient's genetic diversity.Therefore, humanized's transplantable tumor model will be predicted the clinical test results of medicine more exactly, for tumor individual therapy provides more reasonably conceptual design, is a kind of comparatively desirable Gene expression.Jackson Lab (The Jackson Laboratory) of famous American is with every thousands of dollars sell at competitive tumor patient source xenograft tumor mouse models.The Ministry of Industry of France subsidizes the amount of money up to 5,400,000 Euros to this model plan.Therefore, tumor patient source xenograft tumor model oneself formed a huge market.At present, tumor patient source xenograft tumor mouse model, as a kind of novel tumor model, mainly concentrates on breast cancer and lung cancer research, and it is former because liver cancer patient source xenograft tumor mouse model success rate is extremely low.Yan M etc. build liver cancer patient source xenograft tumor mouse model method at Chin J Cancer Res report in 2013: liver cancer tissue is divided into 2mm
3after fritter, directly transplanting is below the omoplate of mouse.The method plantation success rate is only 20.83%.
Therefore, in order to adapt to clinical and scientific research needs, need a kind of new liver cancer patient source xenograft tumor mouse model construction method badly, the method can significantly improve model plantation success rate, constructed model is applicable to drug screening and experimental study, also have manufacturing process simple, lethality is low due to operation simultaneously, and success rate is high, be easy to the feature promoted.
Summary of the invention
First object of the present invention is to overcome the deficiency of existing liver cancer model, provides a kind of animal model that can highly reduce human liver cancer genetics information and biological property, to meet the right demand of medical market.
Another object of the present invention is to provide a kind of construction method of liver cancer patient source xenograft tumor model, significantly improves model plantation success rate.
In order to achieve the above object, the invention provides a kind of liver cancer patient source xenograft tumor mouse model, it is characterized in that, comprise that the back subcutaneous vaccination of forelimb shoulder has the mouse of the liver cancer tissue piece of processing.
Preferably, described mouse is non-obese diabetic severe combined immunodeficiency mouse.
The method for building up that the present invention also provides above-mentioned liver cancer patient to originate xenograft tumor mouse model, it is characterized in that, concrete steps comprise: get fresh HCC tissue, it is processed, liver cancer tissue piece after treatment is inoculated in without knurl mouse forelimb shoulder back subcutaneous, conventional raising, obtains liver cancer patient source xenograft tumor mouse model.
Preferably, the processing method of described fresh HCC tissue comprises: fresh HCC tissue is put into containing the test tube of culture fluid and moved to super-clean bench, and for subsequent use; Taking-up fresh HCC tissue, with culture fluid flushing 2-3 time, being divided into volume is 8-32mm
3liver cancer tissue piece, at 1-10 DEG C of submergence liver cancer tissue piece 30-90 minute, obtain liver cancer tissue piece after treatment with aseptic Incubating Solution.
More preferably, described culture fluid is DMEM culture fluid, its preparation method is: the DMEM of 90 parts by volume is mixed with the hyclone of 10 parts by volume, add penicillin and streptomycin, obtain DMEM culture fluid, wherein, the final concentration of penicillin is 50-200U/ml, and the final concentration of streptomycin is 50-200U/ml.
More preferably, the preparation method of described aseptic Incubating Solution is: by the DMEM of 45 parts by volume, the Matrigel glue of the hyclone of 5 parts by volume and 50 parts by volume mixes, add epidermal growth factor (EGF), fibroblast growth factor (bFGF), penicillin and streptomycin, obtain aseptic Incubating Solution, wherein, the final concentration of epidermal growth factor is 5-20ng/ml, the final concentration of fibroblast growth factor is 5-20ng/ml, the final concentration of penicillin is 50-200U/ml, and the final concentration of streptomycin is 50-200U/ml.
Compared with prior art, the invention has the beneficial effects as follows:
The forelimb shoulder back that patient's liver cancer tissue is directly seeded in experiment mice by the present invention is subcutaneous, retain tumor microenvironment, the correlated characteristic verily reducing in its corresponding patient, whole model process of establishing is simple, become knurl incidence to reach 40%-50%, can be used for liver-cancer medicine screening and liver cancer Mechanism Study.It is simple that the liver cancer patient source xenograft tumor model of setting up by the present invention has manufacturing process, and lethality is low due to operation, and success rate is high, be easy to promote; Can large quantities of making and the good advantage of synchronism, be applicable to drug screening and experimental study.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Test method in following embodiment, if no special instructions, is conventional method.Test material used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.
Same life is divided into tetra-groups of A, B, C, D 3-4 week, 120 male non-obese diabetic severe combined immunodeficiency mouse, and every group of quantity is 30.
Embodiment 1
A kind of liver cancer patient source xenograft tumor mouse model has the mouse of the liver cancer tissue piece of processing to form by the back subcutaneous vaccination of forelimb shoulder, and its method for building up is:
(1) preparation culture fluid: the DMEM of 90 parts by volume is mixed with the hyclone of 10 parts by volume, add penicillin and streptomycin, obtain DMEM culture fluid, wherein, the final concentration of penicillin is 100U/ml, and the final concentration of streptomycin is 100U/ml, obtains culture fluid.
(2) prepare aseptic Incubating Solution: by the DMEM of 45 parts by volume, Matrigel glue (the BD company of the hyclone of 5 parts by volume and 50 parts by volume; Article No.: 356234) mix, add epidermal growth factor (EGF) (Gibco company; Article No.: PHG0314), fibroblast growth factor (bFGF) (Gibco company; Article No.: PHG0264), penicillin and streptomycin, obtain aseptic Incubating Solution, wherein, the final concentration of epidermal growth factor is 10ng/ml, the final concentration of fibroblast growth factor is 10ng/ml, the final concentration of penicillin is 100U/ml, and the final concentration of streptomycin is 100U/ml, obtains aseptic Incubating Solution.
(3) respectively the fresh HCC tissue (in vitro 5 minutes) of 10 corrective surgeries excision is inserted in the test tube containing culture fluid of preparing in (1) in steps, put into ice and transfer to rapidly super-clean bench, for subsequent use;
(4) under aseptic condition, the culture fluid of preparation in step for tumor tissues (1) is rinsed 2 times; Remove slough, select euangiotic liver cancer tissue to be divided into 2 × 2 × 2mm
3fritter, the aseptic Incubating Solution of preparing by step (2) 4 DEG C infiltrate 30 minutes, for subsequent use;
(5) select A group totally 30 male, 3 week age, weight 18-22g, non-obese diabetic severe combined immunodeficiency mouse (NOD/SCID).Each patient's liver cancer tissue is got three fritters, and the forelimb shoulder back that is seeded in respectively 3 mouse is subcutaneous, and concrete inoculation method is: at mouse web portion preserved skin, then with iodophor disinfection.With No. 20 trochars by total amount 2 × 2 × 2mm
3liver cancer tissue thrust in belly midaxillary line place, syringe needle traveling is between skin and peritonaeum, it is subcutaneous that tissue is sent into forelimb shoulder back the most at last, compressing wound is to without hemorrhage;
(6) conventional raising 60 days under SPF level environment, complete and set up liver cancer patient source xenograft tumor model, and be No. 1-10 (each numbering be more than or equal to 1 in 3 mouse of correspondence and become knurl, assert this numbering model modeling success) to pattern number.
A group mouse inoculation is observed mouse general activity and nutritional status etc. after transplanting.Within every three days, grow (L) short (W) footpath mean value, by volume formula V=1/2 (LW with vernier caliper measurement transplantable tumor
2) calculating transplantable tumor average external volume.Transplant the visible local tumor nodule that forms of naked eyes after approximately 4 weeks, within the 10th week, tumour is hemispherical, the average about 1.59cm of tumor volume
3.Dissect mouse, transplantable tumor tissue is flesh of fish sample substantially, and blood is for abundant, and central authorities are visible downright bad, after H & E dyeing, confirm that pathological is hepatocellular carcinoma, consistent with corresponding patient's pathological.Through above-mentioned steps, in No. 2 corresponding 3 mouse of model, there are 2 to become knurls, No. 4 model has 2 to become knurls, and No. 8 and No. 9 models have respectively 1 to become knurl.Finally set up 4 models, the tumor formation rate of mouse is 40%.
Embodiment 2
A kind of liver cancer patient source xenograft tumor mouse model has the mouse of the liver cancer tissue piece of processing to form by the back subcutaneous vaccination of forelimb shoulder, and its method for building up is:
(1) preparation culture fluid: the DMEM of 90 parts by volume is mixed with the hyclone of 10 parts by volume, add penicillin and streptomycin, obtain DMEM culture fluid, wherein, the final concentration of penicillin is 100U/ml, and the final concentration of streptomycin is 100U/ml, obtains culture fluid.
(2) prepare aseptic Incubating Solution: by the DMEM of 45 parts by volume, Matrigel glue (the BD company of the hyclone of 5 parts by volume and 50 parts by volume; Article No.: 356234) mix, add epidermal growth factor (EGF) (Gibco company; Article No.: PHG0314), fibroblast growth factor (bFGF) (Gibco company; Article No.: PHG0264), penicillin and streptomycin, obtain aseptic Incubating Solution, wherein, the final concentration of epidermal growth factor is 10ng/ml, the final concentration of fibroblast growth factor is 10ng/ml, the final concentration of penicillin is 100U/ml, and the final concentration of streptomycin is 100U/ml, obtains aseptic Incubating Solution.
(3) respectively the fresh HCC tissue (in vitro 5 minutes) of 10 corrective surgeries excision is inserted in the test tube containing culture fluid of preparing in (1) in steps, put into ice and transfer to rapidly super-clean bench, for subsequent use;
(4) under aseptic condition, the culture fluid of preparation in step for tumor tissues (1) is rinsed 3 times; Remove slough, select euangiotic liver cancer tissue to be divided into 2 × 2 × 2mm
3fritter, the aseptic Incubating Solution of preparing by step (2) 4 DEG C infiltrate 40 minutes, for subsequent use;
(5) select B group totally 30 male, 3 week age, weight 18-22g, non-obese diabetic severe combined immunodeficiency mouse (NOD/SCID).Each patient's liver cancer tissue is got three fritters, and the forelimb shoulder back that is seeded in respectively 3 mouse is subcutaneous, and concrete inoculation method is: at mouse web portion preserved skin, then with iodophor disinfection.With No. 20 trochars by total amount 2 × 2 × 2mm
3liver cancer tissue thrust in belly midaxillary line place, syringe needle traveling is between skin and peritonaeum, it is subcutaneous that tissue is sent into forelimb shoulder back the most at last, compressing wound is to without hemorrhage;
(6) conventional raising 60 days under SPF level environment, complete and set up liver cancer patient source xenograft tumor model, and be No. 1-10 (each numbering be more than or equal to 1 in 3 mouse of correspondence and become knurl, assert this numbering model modeling success) to pattern number.
B group mouse inoculation is observed mouse general activity and nutritional status etc. after transplanting.Within every three days, grow (L) short (W) footpath mean value, by volume formula V=1/2 (LW with vernier caliper measurement transplantable tumor
2) calculating transplantable tumor average external volume.Transplant the visible local tumor nodule that forms of naked eyes after approximately 4 weeks, within the 11st week, tumour is hemispherical, the average about 1.67cm of tumor volume
3.Dissect mouse, transplantable tumor tissue is flesh of fish sample substantially, and blood is for abundant, and central authorities are visible downright bad, after H & E dyeing, confirm that pathological is hepatocellular carcinoma, consistent with corresponding patient's pathological.Through above-mentioned steps, in No. 1 corresponding 3 mouse of model, there are 2 to become knurl, No. 2 model has 2 to become knurl, and No. 6 model has 2 to become knurl, and No. 9 model has 1 to become knurl.Finally set up 4 models, the tumor formation rate of mouse is 40%.
Embodiment 3
A kind of liver cancer patient source xenograft tumor mouse model has the mouse of the liver cancer tissue piece of processing to form by the back subcutaneous vaccination of forelimb shoulder, and its method for building up is:
(1) preparation culture fluid: the DMEM of 90 parts by volume is mixed with the hyclone of 10 parts by volume, add penicillin and streptomycin, obtain DMEM culture fluid, wherein, the final concentration of penicillin is 100U/ml, and the final concentration of streptomycin is 100U/ml, obtains culture fluid.
(2) prepare aseptic Incubating Solution: by the DMEM of 45 parts by volume, Matrigel glue (the BD company of the hyclone of 5 parts by volume and 50 parts by volume; Article No.: 356234) mix, add epidermal growth factor (EGF) (Gibco company; Article No.: PHG0314), fibroblast growth factor (bFGF) (Gibco company; Article No.: PHG0264), penicillin and streptomycin, obtain aseptic Incubating Solution, wherein, the final concentration of epidermal growth factor is 10ng/ml, the final concentration of fibroblast growth factor is 10ng/ml, the final concentration of penicillin is 100U/ml, and the final concentration of streptomycin is 100U/ml, obtains aseptic Incubating Solution.
(3) respectively the fresh HCC tissue (in vitro 5 minutes) of 10 corrective surgeries excision is inserted in the test tube containing culture fluid of preparing in (1) in steps, put into ice and transfer to rapidly super-clean bench, for subsequent use;
(4) under aseptic condition, the culture fluid of preparation in step for tumor tissues (1) is rinsed 3 times; Remove slough, select euangiotic liver cancer tissue to be divided into 2 × 3 × 3mm
3fritter, the aseptic Incubating Solution of preparing by step (2) 4 DEG C infiltrate 60 minutes, for subsequent use;
(5) select C group totally 30 male, 3 week age, weight 18-22g, non-obese diabetic severe combined immunodeficiency mouse (NOD/SCID).Each patient's liver cancer tissue is got three fritters, and the forelimb shoulder back that is seeded in respectively 3 mouse is subcutaneous, and concrete inoculation method is: at mouse web portion preserved skin, then with iodophor disinfection.With No. 20 trochars by total amount 2 × 3 × 3mm
3liver cancer tissue thrust in belly midaxillary line place, syringe needle traveling is between skin and peritonaeum, it is subcutaneous that tissue is sent into forelimb shoulder back the most at last, compressing wound is to without hemorrhage;
(6) conventional raising 60 days under SPF level environment, complete and set up liver cancer patient source xenograft tumor model, and be No. 1-10 (each numbering be more than or equal to 1 in 3 mouse of correspondence and become knurl, assert this numbering model modeling success) to pattern number.
C group mouse inoculation is observed mouse general activity and nutritional status etc. after transplanting.Within every three days, grow (L) short (W) footpath mean value, by volume formula V=1/2 (LW with vernier caliper measurement transplantable tumor
2) calculating transplantable tumor average external volume.Transplant the visible local tumor nodule that forms of naked eyes after approximately 5 weeks, within the 10th week, tumour is hemispherical, the average about 1.36cm of tumor volume
3.Dissect mouse, transplantable tumor tissue is flesh of fish sample substantially, and blood is for abundant, and central authorities are visible downright bad, after H & E dyeing, confirm that pathological is hepatocellular carcinoma, consistent with corresponding patient's pathological.Through above-mentioned steps, in No. 2 corresponding 3 mouse of model, there are 2 to become knurl, No. 5 model has 3 to become knurl, and No. 7 model has 1 to become knurl, and No. 10 model has 2 to become knurl.Finally set up 4 models, the tumor formation rate of mouse is 40%.
Embodiment 4
A kind of liver cancer patient source xenograft tumor mouse model has the mouse of the liver cancer tissue piece of processing to form by the back subcutaneous vaccination of forelimb shoulder, and its method for building up is:
(1) preparation culture fluid: the DMEM of 90 parts by volume is mixed with the hyclone of 10 parts by volume, add penicillin and streptomycin, obtain DMEM culture fluid, wherein, the final concentration of penicillin is 100U/ml, and the final concentration of streptomycin is 100U/ml, obtains culture fluid.
(2) prepare aseptic Incubating Solution: by the DMEM of 45 parts by volume, Matrigel glue (the BD company of the hyclone of 5 parts by volume and 50 parts by volume; Article No.: 356234) mix, add epidermal growth factor (EGF) (Gibco company; Article No.: PHG0314), fibroblast growth factor (bFGF) (Gibco company; Article No.: PHG0264), penicillin and streptomycin, obtain aseptic Incubating Solution, wherein, the final concentration of epidermal growth factor is 10ng/ml, the final concentration of fibroblast growth factor is 10ng/ml, the final concentration of penicillin is 100U/ml, and the final concentration of streptomycin is 100U/ml, obtains aseptic Incubating Solution.
(3) respectively the fresh HCC tissue (in vitro 5 minutes) of 10 corrective surgeries excision is inserted in the test tube containing culture fluid of preparing in (1) in steps, put into ice and transfer to rapidly super-clean bench, for subsequent use;
(4) under aseptic condition, the culture fluid of preparation in step for tumor tissues (1) is rinsed 3 times; Remove slough, select euangiotic liver cancer tissue to be divided into 2 × 4 × 4mm
3fritter, the aseptic Incubating Solution of preparing by step (2) 4 DEG C infiltrate 90 minutes, for subsequent use;
(5) select D group totally 30 male, 3 week age, weight 18-22g, non-obese diabetic severe combined immunodeficiency mouse (NOD/SCID).Each patient's liver cancer tissue is got three fritters, and the forelimb shoulder back that is seeded in respectively 3 mouse is subcutaneous, and concrete inoculation method is: at mouse web portion preserved skin, then with iodophor disinfection.With No. 20 trochars by total amount 2 × 4 × 4mm
3liver cancer tissue thrust in belly midaxillary line place, syringe needle traveling is between skin and peritonaeum, it is subcutaneous that tissue is sent into forelimb shoulder back the most at last, compressing wound is to without hemorrhage;
(6) conventional raising 60 days under SPF level environment, complete and set up liver cancer patient source xenograft tumor model, and be No. 1-10 (each numbering be more than or equal to 1 in 3 mouse of correspondence and become knurl, assert this numbering model modeling success) to pattern number.
D group mouse inoculation is observed mouse general activity and nutritional status etc. after transplanting.Within every three days, grow (L) short (W) footpath mean value, by volume formula V=1/2 (LW with vernier caliper measurement transplantable tumor
2) calculating transplantable tumor average external volume.Transplant the visible local tumor nodule that forms of naked eyes after approximately 4 weeks, within the 10th week, tumour is hemispherical, the average about 1.71cm of tumor volume
3.Dissect mouse, transplantable tumor tissue is flesh of fish sample substantially, and blood is for abundant, and central authorities are obviously downright bad, after H & E dyeing, confirms that pathological is hepatocellular carcinoma, consistent with corresponding patient's pathological.Through above-mentioned steps, in No. 1 corresponding 3 mouse of model, there are 3 to become knurls, No. 3, No. 4 and No. 5 models have 2 to become knurls, and No. 10 model has 3 to become knurls.Finally set up 5 models, the tumor formation rate of mouse is 50%.
Claims (6)
1. a liver cancer patient source xenograft tumor mouse model, is characterized in that, comprises that the back subcutaneous vaccination of forelimb shoulder has the mouse of the liver cancer tissue piece of processing.
2. liver cancer patient as claimed in claim 1 source xenograft tumor mouse model, is characterized in that, described mouse is non-obese diabetic severe combined immunodeficiency mouse.
3. the method for building up of liver cancer patient claimed in claim 1 source xenograft tumor mouse model, it is characterized in that, concrete steps comprise: get fresh HCC tissue, it is processed, liver cancer tissue piece after treatment is inoculated in without knurl mouse forelimb shoulder back subcutaneous, conventional raising, obtains liver cancer patient source xenograft tumor mouse model.
4. the method for building up of liver cancer patient as claimed in claim 3 source xenograft tumor mouse model, is characterized in that, the processing method of described fresh HCC tissue comprises: fresh HCC tissue is put into containing the test tube of culture fluid and moved to super-clean bench, and for subsequent use; Taking-up fresh HCC tissue, with culture fluid flushing 2-3 time, being divided into volume is 8-32mm
3liver cancer tissue piece, at 1-10 DEG C of submergence liver cancer tissue piece 30-90 minute, obtain liver cancer tissue piece after treatment with aseptic Incubating Solution.
5. the method for building up of liver cancer patient as claimed in claim 4 source xenograft tumor mouse model, it is characterized in that, described culture fluid is DMEM culture fluid, its preparation method is: the DMEM of 90 parts by volume is mixed with the hyclone of 10 parts by volume, add penicillin and streptomycin, obtain DMEM culture fluid, wherein, the final concentration of penicillin is 50-200U/ml, and the final concentration of streptomycin is 50-200U/ml.
6. the method for building up of liver cancer patient as claimed in claim 4 source xenograft tumor mouse model, it is characterized in that, the preparation method of described aseptic Incubating Solution is: by the DMEM of 45 parts by volume, the Matrigel glue of the hyclone of 5 parts by volume and 50 parts by volume mixes, add epidermal growth factor (EGF), fibroblast growth factor (bFGF), penicillin and streptomycin, obtain aseptic Incubating Solution, wherein, the final concentration of epidermal growth factor is 5-20ng/ml, the final concentration of fibroblast growth factor is 5-20ng/ml, the final concentration of penicillin is 50-200U/ml, the final concentration of streptomycin is 50-200U/ml.
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