CN102766599A - Application of esophageal squamous cell carcinoma primary tumor strain CH-H-1 - Google Patents
Application of esophageal squamous cell carcinoma primary tumor strain CH-H-1 Download PDFInfo
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Abstract
The invention relates to an application of an esophageal squamous cell carcinoma primary tumor strain CH-H-1 in a mammal for producing esophageal squamous cell carcinoma cells. The primary tumor strain CH-H-1 disclosed by the invention can realize multiple passages through NOD/SCID mice, has significance for solving the bottleneck that the current treatment of the esophageal squamous cell carcinoma can not avoid distant metastasis, and is an ideal object for fundamental research on the esophageal squamous cell carcinoma and clinical early application.
Description
Technical field
The invention belongs to former generation knurl xenotransplantation tumor model field, the application of particularly a kind of esophageal squamous cell carcinoma knurl strain of former generation CH-H-1.
Background technology
For a long time, all human tumor preclinical studies are defined to the vitro culture of tumour cell or studying at body of lotus knurl animal pattern.It is found that in practice; Most human tumor cell can not long-term surviving after the living environment in having broken away from original body; Only there be the tumour cell of few part behind environmental screening can be at the external growth of going down to posterity for a long time (World Cancer report2008.IARC Press; Lyon, 2008).Since George Gey has set up the external SCC cell strain HeLa cell strain that goes down to posterity of human first strain first in nineteen fifty-two; The tumor cell line model is because of going down to posterity in external long-term stability; Repeatability advantage such as strong and obtain expert's generally favor, the tumor cell line that makes up of success has almost been contained the tumor tissues of all kinds.In the last thirty years; The breakthrough of nearly all tumor research and the exploitation of antitumor drug all stem from this application of model (Voskoglou-Nomikos T; Pater JL; Seymour L.Clinical predictive value ofthe in vitro cell line, human xenograft, and mouse allograft preclinical cancer models.Clin Cancer Res2003; 9 (11): 4227-39).
Yet Along with people's deepens continuously to tumour character and tumor cell line model research, and the existing deficiency of this model self also day by day is familiar with by people.It should be noted that often have some antitumor drugs of once in preclinical study, obtaining ideal treatment effect in the later stage clinical verification barely satisfactory during this time, in addition with early stage the result far from each other.As studying epidermal growth factor recipient tyrosine kinase inhibitor Iressa (Iressa; Have another name called lucky expense for Buddhist nun Gefitinib) it is found that in the process to the nonsmall-cell lung cancer therapeutic action; Preclinical study obvious curative effect Iressa is to not remarkable (the Lynch TJ of most of nonsmall-cell lung cancer patient curative effects; Bell DW, Sordella R et al.Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib.N Engl J Med2004; 350 (21): 2129-39), (Paez JG, Janne PA, Lee JC et al.EGFR mutations in lung cancer:correlation with clinical response to gefitinib therapy.Science2004; 304 (5676): 1497-500), (Sordella R, Bell DW, Haber DA et al.Gefitinib-sensitizing EGFR mutations in lung cancer activate anti-apoptotic pathways.Science2004; 305 (5687): 1163-7).Although the later stage has research to show; Iressa is evident in efficacy to the nonsmall-cell lung cancer patient of some EGFR transgenation; But the R&D process in its early stage clearly illustrates that the medicine by tumor cell line and transplanted tumor structure thereof that widely adopts at present sieves between platform and the clinical practice result of treatment and also exists evident difference.
Recent study finds that tumor tissues is that the different tumour cell of do as one likes matter is formed, and its one-tenth knurl and energy for growth separately all exists evident difference.Like discovery (Shmelkov SV such as Shmelkov; Butler JM; Hooper AT et al.CD133expression is not restricted to stem cells, and both CD133+and CD133-metastatic colon cancer cells initiate tumors.J Clin Invest2008; 118 (6): 2111-20), in colorectal carcinoma hepatic metastases knurl, the CD133 positive tumor cell can break up that to produce aggressive stronger, the negative colon cancer cell of the CD133 that tumor formation rate is higher.Owing to tumor cell line is normally got by the amplification of minority or even one tumour cell, so the cell strain model often reflects is the character of part tumour cell.Particularly importantly; Increasing research shows; Tumour cell reaches the ability that goes down to posterity for a long time in order to obtain external adherent growth in building the strain process; Its cell function and inherited character all can be adjusted accordingly, and cause some important biological characteristics to change therefrom, comprising: the unlatching of genetic expression or close (Pandita A; Aldape KD, Zadeh G et al.Contrasting in vivo and in vitro fates of glioblastoma cell subpopulations with amplified EGFR.Genes Chromosomes Cancer2004; 39 (1): 29-36), (De Witt Hamer PC; Van Tilborg AA, Eijk PP et al.The genomic profile of human malignant glioma is altered early in primary cell culture and preserved in spheroids.Oncogene2008; 27 (14): 2091-6); Migration and transfer ability change (Vescovi AL, Galli R, Reynolds BA.Brain tumour stem cells.Nat Rev Cancer2006; 6 (6): 425-36); Change of tumor stem cell crowd proportional imbalance and signal transduction pathway or the like (Clement V; SanchezP; De Tribolet N et al.HEDGEHOG-GLI1 signaling regulates human glioma growth; Cancer stem cell self-renewal, and tumorigenicity.Curr Biol2007; 17 (2): 165-72), (Sasai K; Romer JT, Lee Y et al.Shh pathway activity is down-regulated in cultured medulloblastoma cells:implications for preclinical studies.Cancer Res2006; 66 (8): 4215-22).Further research shows; Even the tumor cell line cell is implanted immunodeficient mouse and is recovered its three dimensional growth form; Therefore above-mentioned change can not obtain correcting (Daniel VC; Marchionni L, Hierman JS et al.A primary xenograft model of small-cell lung cancer reveals irreversible changes in gene expression imposed by culture in vitro.Cancer Res2009; 69 (8): 3364-73).Can infer, the tumor cell line model with former generation tumour qualitative different, certainly will cause the serious disconnection between antitumor drug preclinical study and the actual clinical result of treatment.
In order to overcome the existing deficiency of tumor cell line model, there is the scholar once once attempting setting up mouse source tumour in the phantom type and attempt the lethal effect of mouse source tumour cell to be inferred that it is to people source tumor treatment effect through observing antitumor drug.But follow-up discovers; Identical antitumor drug differs totally different to people source and mouse source tumor treatment effect, and mouse source tumor model is to the suggesting effect of clinical therapeutic efficacy even be worse than traditional tumor cell line model (Teicher BA.Human tumor xenografts and mouse models of human tumors:re-discovering the models.Expert Opin Drug Discov2009; 11), (Richmond A, Su Y.Mouse xenograft models vs GEM models for human cancer therapeutics.Dis Model Mech2008 4 (12):; 1 (2-3): 78-82).For this reason; Some scholars look for another way; Employing is directly implanted immunodeficient mouse construct in vitro knurl xenotransplantation of former generation tumor model (Morton CL, Houghton PJ.Establishment of human tumor xenografts in immunodeficient mice.Nat Protoc2007 with the fresh tumor tissues of human body; 2 (2): 247-50), (Sausville EA, Burger AM.Contributions of human tumor xenografts to anticancer drug development.Cancer Res2006; 66 (7): 3351-4, discussion4).Preliminary result of study shows that the former generation knurl xenotransplantation tumor model that makes up under the three dimensional growth condition can be simulated tumour cell better at the intravital actual upgrowth situation of people.
Former generation knurl xenotransplantation tumor model can not only reflect tumour cell better in the intravital growth characteristics of people, and can keep go down to posterity under the stable prerequisite of proterties amplification and prolonged preservation.This shows; Set up former generation knurl xenotransplantation tumor model and not only help to deepen understanding and understanding the tumor growth rule; And being expected to take this to set up the screening anti-tumor medicine platform of more pressing close to the clinical practice result of treatment, its importance in following tumor research is self-evident.
Summary of the invention
Technical problem to be solved by this invention provides the application of a kind of esophageal squamous cell carcinoma knurl strain of former generation CH-H-1; This former generation knurl strain CH-H-1 can repeatedly go down to posterity through the NOD/SCID mouse; For solving the bottleneck problem that to avoid in this present esophageal squamous cell carcinoma treatment of metastasis; Significant, be the desirable object that esophageal squamous cell carcinoma fundamental research and preclinical phase are used.
The application of a kind of esophageal squamous cell carcinoma knurl strain of former generation CH-H-1 of the present invention is used for producing the esophageal squamous cell carcinoma cell Mammals.
Said former generation knurl strain CH-H-1 also comprises the filial generation tumor tissues of former generation knurl strain.
Said Mammals is a mouse.
Said mouse is the NOD/SCID mouse.
The preparation method of said former generation knurl strain CH-H-1 and filial generation tumor tissues thereof comprises:
(1) NOD/SCID mouse 1d before inoculation accepts the pre-treatment of cobalt 60-gamma-ray irradiation, and irradiation dose is 2.5Gy/;
(2) abdominal injection vetanarcol anesthetized mice, with former generation esophageal squamous cell carcinoma tissue be cut into small tissue blocks, subcutaneous vaccination mouse in back produces first-generation transplanted tumor, tumor formation rate 100%;
(3) after first-generation transplanted tumor diameter surpasses 10mm, put to death tumor-bearing mice; Tumor tissue is cut into small pieces and subcutaneous vaccination NOD/SCID mouse once more, produces s-generation transplanted tumor, all the other transplanted tumors are organized freezing preservation, repeat this process until the 40th generation transplanted tumor form and carry out character and identify freezing preservation.
Beneficial effect
(1) esophageal squamous cell carcinoma of the present invention knurl strain of former generation proterties is stable, can repeatedly go down to posterity through the NOD/SCID mouse;
(2) esophageal squamous cell carcinoma of the present invention knurl strain of former generation has the biological character of esophageal squamous cell carcinoma clinically, possesses the characteristic of high metastatic potential.
For solving the bottleneck problem that can't avoid in this present esophageal squamous cell carcinoma treatment of metastasis, significant, be the desirable object that esophageal squamous cell carcinoma fundamental research and preclinical phase are used.
Description of drawings
Fig. 1 for former generation esophageal squamous cell carcinoma (left side) and the 40th generation knurl strain transplanted tumor (right side) tissue general form HE dyeing (a), p75 (NTR) (b), the comparison diagram of p63 (c), β 1-integrin (d) and β 4-integrin (e);
Fig. 2 is the tissue paraffin section de of former generation esophageal squamous cell carcinoma (left side) and the 40th generation transplanted tumor (right side), the Ki-67 immunohistochemical staining;
Fig. 3 is the tissue paraffin section de of former generation esophageal squamous cell carcinoma (left side) and the 40th generation transplanted tumor (right side), the PCNA immunohistochemical staining;
Fig. 4 is the proliferation index that former generation esophageal squamous cell carcinoma (left side) and the 40th generation transplanted tumor (right side) detect through flow cytometer;
Fig. 5 is the apoptotic index that former generation esophageal squamous cell carcinoma (left side) and the 40th generation transplanted tumor (right side) detect through flow cytometer.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.Should be understood that in addition those skilled in the art can do various changes or modification to the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Embodiment 1
1. the foundation of former generation esophageal squamous cell carcinoma xenotransplantation tumour knurl strain
(1) laboratory animal: the NOD/SCID mouse, in age in 4-6 week, male and female are regardless of, available from the The 2nd Army Medical College Experimental Animal Center.
(2) tumor tissues: the fresh esophageal neoplasm tissue sample that is taken from 53 years old male sex's III a phase esophageal squamous cell carcinoma patient of 1 example that attached Changhai hospital of The 2nd Army Medical College cardiothoracic surgery undergos surgery in January, 2007.
(3) former generation knurl xenotransplantation becomes knurl and goes down to posterity amplification:
1. NOD/SCID mouse 1d before inoculation accepts the pre-treatment of cobalt 60-gamma-ray irradiation, and irradiation dose is 2.5Gy/.
2. abdominal injection vetanarcol (0.05mg/g body weight) anesthetized mice, former generation esophageal squamous cell carcinoma tissue be cut into the small tissue blocks of about 3 * 3 * 3mm, subcutaneous vaccination mouse in back produces the first-generation (F1) transplanted tumor, tumor formation rate 100%.
3. after F1 transplanted tumor diameter surpasses 10mm, put to death tumor-bearing mice.Tumor tissue is cut into fritter and the subcutaneous vaccination NOD/SCID mouse once more of about 3 * 3 * 3mm, produces the s-generation (F2) transplanted tumor, all the other transplanted tumors are organized freezing preservation.Repeat this process until the 40th generation (F40) transplanted tumor form and carry out character and identify.
(4) the transplanted tumor profound hypothermia is preserved and recovery back one-tenth knurl:
1. get the transplanted tumor tissue that respectively goes down to posterity, it is cut into the small tissue blocks of about 1 * 1 * 1mm, DMEM substratum rinsing tissue, the centrifugal 5min of 500rpm abandons supernatant.Tissue block added protection liquid (the DMEM substratum that contains 20% foetal calf serum and 10%DMSO) in 1: 1 by volume, was sub-packed in behind the mixing in a plurality of frozen pipes.Frozen pipe is in 4 ℃ of precooling 10min, and with being placed in the program control cooling instrument and by following programmed cooling: 4 ℃ are initial, and-0.5 ℃/min is to-30 ℃, subsequently-5 ℃/min is to-100 ℃, and take out frozen pipe and place liquid nitrogen container to preserve.
2. the transplanted tumor tissue freezing 1,6, and 12; Take out F1, F10, F20, F30, each frozen pipe of F40 after 24 months respectively; Thaw rapidly in 37 ℃ of water-baths, behind the DMEM substratum cleansing tissue, 500rpm is centrifugal, and 5min removes supernatant; Get tissue block subcutaneous vaccination NOD/SCID mouse according to preceding method, the more different frozen times are organized into the influence of knurl ability to transplanted tumor.The present invention find until the 40th generation (F40) frozen 24 months knurl strain tumor formation rate with former generation knurl strain and other respectively for knurl strain tumor formation rate indistinction, be 100%.
2. the character of former generation esophageal squamous cell carcinoma xenotransplantation tumour knurl strain is identified
(1) knurl strain phenotype analytical
Get former generation esophageal squamous cell carcinoma and the tissue of corresponding F1, F10, F20, F30, F40 transplanted tumor, fixing back row routine paraffin wax embedded section, HE dyeing is different sources tumor tissue general form relatively; P75 (NTR), p63, involucrin, β 1-integrin and β 4-integrin immunohistochemical staining be protein expression level and positive cell distribution situation relatively.The result confirm respectively to go down to posterity the knurl strain phenotype analytical and former generation tumour do not have significant difference.(Fig. 1)
(2) knurl strain multiplication capacity is analyzed
1. get former generation esophageal squamous cell carcinoma and the tissue paraffin section de of corresponding F1, F5, F10, F15, F20, F30, F40 transplanted tumor, Ki-67, PCNA dyeing is proliferative cell ratio in the different sources tumor tissue relatively; TUNEL dyeing comparison of tumor apoptosis rate.Result's knurl strain that confirms respectively to go down to posterity does not have significant difference in proliferative cell ratio, apoptosis of tumor cells rate.(Fig. 2, Fig. 3)
2. get former generation esophageal squamous cell carcinoma and the flesh tissue of corresponding F1, F10, F20, F30, F40 transplanted tumor; It is cut into the small tissue blocks of about 1 * 1 * 1mm; IV Collagen Type VI enzyme/mucinase enzymic digestion obtains single cell suspension, detects the proliferation index (cell cycle analysis method) and the apoptotic index (the two mark methods of AnnexinV/7-AAD) of different sources tumour cell through flow cytometer.Result's knurl strain that confirms respectively to go down to posterity does not have significant difference at proliferation index, apoptotic index.(Fig. 4, Fig. 5)
Above result shows that the former generation xenotransplantation of esophageal squamous cell carcinoma knurl strain CH-H-1 can not only reflect tumour cell better in the intravital growth characteristics of people, and can keep go down to posterity under the stable prerequisite of proterties amplification and prolonged preservation.The foundation of this knurl strain not only helps to deepen understanding and the understanding to the tumor growth rule, and is expected to take this to set up the screening anti-tumor medicine platform of more pressing close to the clinical practice result of treatment.
Claims (5)
1. the application of esophageal squamous cell carcinoma knurl strain of a former generation CH-H-1 is characterized in that: be used for producing the esophageal squamous cell carcinoma cell Mammals.
2. the application of a kind of esophageal squamous cell carcinoma knurl strain of former generation CH-H-1 according to claim 1 is characterized in that: said former generation knurl strain CH-H-1 also comprises the filial generation tumor tissues of former generation knurl strain.
3. the application of a kind of esophageal squamous cell carcinoma knurl strain of former generation CH-H-1 according to claim 2 is characterized in that: the preparation method of said former generation knurl strain CH-H-1 and filial generation tumor tissues thereof comprises:
(1) NOD/SCID mouse 1d before inoculation accepts the pre-treatment of cobalt 60-gamma-ray irradiation, and irradiation dose is 2.5Gy/;
(2) abdominal injection vetanarcol anesthetized mice, with former generation esophageal squamous cell carcinoma tissue be cut into small tissue blocks, subcutaneous vaccination mouse in back produces first-generation transplanted tumor, tumor formation rate 100%;
(3) after first-generation transplanted tumor diameter surpasses 10mm, put to death tumor-bearing mice; Tumor tissue is cut into small pieces and subcutaneous vaccination NOD/SCID mouse once more, produces s-generation transplanted tumor, all the other transplanted tumors are organized freezing preservation, repeat this process until the 40th generation transplanted tumor form and carry out character and identify freezing preservation.
4. the application of a kind of esophageal squamous cell carcinoma knurl strain of former generation CH-H-1 according to claim 1, it is characterized in that: said Mammals is a mouse.
5. the application of a kind of esophageal squamous cell carcinoma knurl strain of former generation CH-H-1 according to claim 4, it is characterized in that: said mouse is the NOD/SCID mouse.
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| CN110760477A (en) * | 2019-10-10 | 2020-02-07 | 杭州广科安德生物科技有限公司 | Establishment method of mouse liver high-metastasis intestinal cancer cell strain, cell strain and application |
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| CN107541496A (en) * | 2016-06-27 | 2018-01-05 | 安徽省立医院 | A kind of human esophageal carcinoma cell line and its application |
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| CN106011069A (en) * | 2016-07-22 | 2016-10-12 | 浙江省肿瘤医院 | Esophageal cancer cell line and application thereof |
| CN106047815A (en) * | 2016-07-22 | 2016-10-26 | 浙江省肿瘤医院 | Esophageal cancer cell line and application thereof |
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| CN106190981A (en) * | 2016-07-22 | 2016-12-07 | 浙江省肿瘤医院 | A kind of esophageal carcinoma cell line and application thereof |
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| CN106011068A (en) * | 2016-07-22 | 2016-10-12 | 浙江省肿瘤医院 | Esophageal cancer cell line and application thereof |
| CN110760477A (en) * | 2019-10-10 | 2020-02-07 | 杭州广科安德生物科技有限公司 | Establishment method of mouse liver high-metastasis intestinal cancer cell strain, cell strain and application |
| CN113249325A (en) * | 2020-02-11 | 2021-08-13 | 合肥中科普瑞昇生物医药科技有限公司 | Culture medium and culture method of esophageal squamous carcinoma primary cells |
| CN113249325B (en) * | 2020-02-11 | 2023-02-17 | 合肥中科普瑞昇生物医药科技有限公司 | Culture medium and culture method of esophageal squamous carcinoma primary cells |
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Application publication date: 20121107 |