細(xì)胞組織消化常用的幾種酶的選擇

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直接從生物體獲取的組織，一般需要將其消化成單個(gè)細(xì)胞才能進(jìn)行體外培養(yǎng)。這種直接從離體組織獲得的細(xì)胞，更接近于生物體內(nèi)的生活狀態(tài)，且生物性狀尚未發(fā)生很大改變，因此在藥物篩選、細(xì)胞移植、類器官培養(yǎng)、腫瘤研究等眾多領(lǐng)域備受歡迎。但組織消化過程中常遇到多種問題，例如消化不完全、細(xì)胞死亡率高等。如何克服這些問題，其實(shí)消化酶的選擇是關(guān)鍵。
本文將為您介紹一些常用的消化酶及其適用組織，并為您羅列多種正常和腫瘤組織消化方案，供您參考。

來了解組織消化過程中常用的酶吧～

1.胰蛋白酶
胰蛋白酶(Trypsin)是目前應(yīng)用最廣泛的消化試劑，通過作用于與賴氨酸或精氨酸相連接的肽腱，除去細(xì)胞間粘蛋白及糖蛋白，影響細(xì)胞骨架，從而使細(xì)胞分離。胰蛋白酶適用于消化細(xì)胞間質(zhì)較少的軟組織，如胚胎、上皮、肝、腎等組織。但對于纖維組織和較硬的癌組織的效果差，常常將胰蛋白酶與其他消化酶聯(lián)合使用。EDTA可以用來增強(qiáng)胰蛋白酶的水解功能，而血清則會(huì)影響胰蛋白酶的活性。

2.膠原酶
膠原酶(Collagenase)通過水解細(xì)胞間質(zhì)的脯氨酸，從而使細(xì)胞離散。膠原酶對膠原的消化作用很強(qiáng)，它僅對細(xì)胞間質(zhì)有消化作用而對細(xì)胞損害不大，因此適于消化分離纖維性組織和較硬的癌組織。鈣鎂離子和血清不會(huì)對膠原酶的活性及消化作用產(chǎn)生影響。
膠原酶可細(xì)分為Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ型膠原酶，不同類型膠原酶適用范圍不同(詳見下表)。

膠原酶類型及適用范圍表

3.透明質(zhì)酸酶
透明質(zhì)酸酶(Hyaluronidase)通過隨機(jī)降解透明質(zhì)酸的β-N-乙酰己糖胺-[1-4]糖苷鍵，從而降低透明質(zhì)酸的活性，常與膠原酶等聯(lián)合使用，用于結(jié)締組織的分離。

4.脫氧核糖核酸酶Ⅰ
脫氧核糖核酸酶Ⅰ(DeoxyribonucleaseⅠ, DNaseⅠ)作用于細(xì)胞分離降解出的DNA，防止DNA導(dǎo)致的細(xì)胞凝集，對細(xì)胞完整性無破壞作用。通常配合膠原酶或透明質(zhì)酸酶等聯(lián)合使用，不會(huì)單獨(dú)使用。

5.中性蛋白酶
中性蛋白酶(Dispase)具有溫和的蛋白酶水解活性，不會(huì)損壞細(xì)胞膜的完整性，常與膠原酶等聯(lián)合使用，其分離纖維樣組織的效率高于上皮組織。

6.彈性蛋白酶
彈性蛋白酶(Elastase)通過作用于彈性蛋白的肽鍵、酰胺鍵和酯鍵將其水解，常與胰蛋白酶和膠原酶等聯(lián)合使用，用于分離結(jié)締組織和含有大量細(xì)胞網(wǎng)狀纖維的組織。

還有消化方案供您參考～
不同組織消化方案有所不同，同種組織的消化方案也不盡相同，考慮到組織的特殊性及酶的多樣性，針對具體組織仍需試驗(yàn)摸索以確定最佳消化方案。以下列表方法供大家參考。

表一：正常組織消化用酶列表

表二：腫瘤組織消化用酶列表

參考文獻(xiàn)

1. Kasai-Brunswick TH., et al., (2017) Cardiosphere-derived Cells Do Not Improve Cardiac Function in Rats With Cardiac Failure. Stem Cell Res Ther. Feb 15;8(1):36.
2. Huch.M., et al., (2015) Long-term Culture of Genome-Stable Bipotent Stem Cells From Adult Human Liver. Cell. Jan 15;160(1-2):299-312.
3. Holt PG., et al., (1986) Extraction of Immune and Inflammatory Cells From Human Lung Parenchyma: Evaluation of an Enzymatic Digestion Procedure. Clin Exp Immunol. Oct;66(1):188-200.
4. Campbell AM., et al., (1993) Modulation of Eicosanoid and Histamine Release From Human Dispersed Lung Cells by Terfenadine. Allergy. Feb;48(2):125-9.
5. Sanchez-Romero N., et al., (2020) A Simple Method for the Isolation and Detailed Characterization of Primary Human Proximal Tubule Cells for Renal Replacement Therapy. Int J Artif Organs. Jan;43(1):45-57.
6. Ataollahi F., et al., (2014) New Method for the Isolation of Endothelial Cells From Large Vessels. Cytotherapy. Aug;16(8):1145-52.
7. Leelatian N., et al., (2017) Single cell analysis of human tissues and solid tumors with mass cytometry. Cytometry B Clin Cytom. Jan;92(1):68-78.
8. Roberts EG., et al., (2019) Evaluation of Placental Mesenchymal Stem Cell Sheets for Myocardial Repair and Regeneration. Tissue Eng Part A Jun;25(11-12):867-877.
9. Jankovic-Karasoulos T., et al., (2018) Isolation of villous cytotrophoblasts from second trimester human placentas.
Placenta. Dec 15;74:55-58.
10. Tang YL., et al., (2007) A Novel Two-Step Procedure to Expand Cardiac Sca-1+ Cells Clonally. Biochem Biophys Res Commun. Aug 10;359(4):877-83.
11. Meyer J. ., et al., (2016) An optimized method for mouse liver sinusoidal endothelial cell isolation.
Exp Cell Res. Dec 10;349(2):291-301.
12. Nakano H., et al., 1799 (2018) Isolation and Purification of Epithelial and Endothelial Cells from Mouse Lung. Methods Mol Biol.:59-69.
13. Jain R. ., et al., (2014) Isolation of thymic epithelial cells and analysis by flow cytometry. Curr Protoc Immunol. Nov 3;107:3.26.1-3.26.15.
14. Tran LS., et al., (2018) Isolation of Mouse Primary Gastric Epithelial Cells to Investigate the Mechanisms of Helicobacter Pylori Associated Disease. Methods Mol Biol. 1725:119-126.
15. De Clercq K., et al., (2017) Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization. J Vis Exp Mar 2;(121):55168.
16. Sharon, Y., et al., (2013) Isolation of Normal and Cancer-Associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS). J Vis Exp 71, e4425.
17. Zhang Q., et al., (2016) Isolation and Culture of Single Cell Types from Rat Liver. Cells Tissues Organs. 201(4):253-67.
18. Al-Eisa A., (2017) IgA Enhances IGF-1 Mitogenic Activity Via Receptor Modulation in Glomerular Mesangial Cells: Implications for IgA-Induced Nephropathy. Kidney Blood Press Res.;42(3):391-397.
19. Githens S., et al., (1994) Isolation and Culture of Rhesus Monkey Pancreatic Ductules and Ductule-Like Epithelium Pancreas. Jan;9(1):20-31.
20. Caperna TJ., et al., (2011) Culture of porcine hepatocytes or bile duct epithelial cells by inductive serum-free media In Vitro Cell Dev Biol Anim. Mar;47(3):218-33.
21. Ataollahi F., et al., (2014) New method for the isolation of endothelial cells from large vessels. Cytotherapy. Aug;16(8):1145-52.
22. Widowati W., et al., (2018) Isolation, Characterization and Proliferation of Cancer Cells from Breast Cancer Patients. Acta Inform Med. Dec;26(4):240-244.
23. Beaupain R., et al., (1993) “Normal” breast cells adjacent to a tumor grown in long-term three-dimensional culture. In Vitro Cellular & Developmental Biology – Plant, 29(2): 100-104.
24. Leung C K., et al., (1982) Morphological and proliferative characteristics of human breast tumor cells cultured on plastic and in collagen matrix. In Vitro Cellular & Developmental Biology – Plant, 18(5): 476-482.
25. Chou J., et al., (2013) Phenotypic and Transcriptional Fidelity of Patient-Derived Colon Cancer Xenografts in Immune-Deficient Mice. PLOS ONE, 8(11).
26. Friedman E., et al., (1981) Tissue culture of human epithelial cells from benign colonic tumors. In Vitro Cellular & Developmental Biology – Plant, 17(7): 632-644.
27. Brattain M G., et al., (1983) Characterization of human colon carcinoma cell lines isolated from a single primary tumour. British Journal of Cancer, 47(3): 373-381.
28. Quatromoni J G., et al., (2015) An optimized disaggregation method for human lung tumors that preserves the phenotype and function of the immune cells. Journal of Leukocyte Biology, 97(1): 201-209.
29. Zhuang X., et al., (2015) Identification of novel vascular targets in lung cancer Br J Cancer. Feb 3; 112(3): 485–494.
30. Welte Y., et al., (2013) Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma. Journal of Visualized Experiments.
31. Tillotson LG., et al., (2001) Isolation, Maintenance, and Characterization of Human Pancreatic Islet Tumor Cells Expressing Vasoactive Intestinal Peptide Pancreas. Jan;22(1):91-8.
32. Nakashiro K., et al., (2004) Phenotypic Switch from Paracrine to Autocrine Role of Hepatocyte Growth Factor in an Androgen-Independent Human Prostatic Carcinoma Cell Line, CWR22R. American Journal of Pathology, 165(2): 533-540.
33. Sheela S., et al., (1990) Angiogenic and invasive properties of neurofibroma Schwann cells. Journal of Cell Biology, 111(2): 645-653.
34. Sacks P G., et al., (1988) Establishment and characterization of two new squamous cell carcinoma cell lines derived from tumors of the head and neck. Cancer Research, 48(10): 2858-2866.
35. Kim M P., et al., (2009) Orthotopic and heterotopic generation of murine pancreatic cancer xenografts [J]. Nature Protocols, 4(11): 1670-1680.
36. Rasheed Z A., et al., (2010) Isolation of Stem Cells from Human Pancreatic Cancer Xenografts. Journal of Visualized Experiments.
37. Vaughan A E., et al., (2011) Lung Cancer in Mice Induced by the Jaagsiekte Sheep Retrovirus Envelope Protein Is Not Maintained by Rare Cancer Stem Cells, but Tumorigenicity Does Correlate with Wnt Pathway Activation. Molecular Cancer Research, 10(1): 86-95.
38. Liu X., et al., (2013) Nonlinear Growth Kinetics of Breast Cancer Stem Cells: Implications for Cancer Stem Cell Targeted Therapy. Scientific Reports , 3(1): 2473-2473.
39. Kazerounian S., et al., (2013) RhoB differentially controls Akt function in tumor cells and stromal endothelial cells during breast tumorigenesis. Cancer Research, 73(1): 50-61.
40. Mazzoleni S., et al., (2013) Gene Signatures Distinguish Stage-Specific Prostate Cancer Stem Cells Isolated From Transgenic Adenocarcinoma of the Mouse Prostate Lesions and Predict the Malignancy of Human Tumors. Stem Cells Translational Medicine, 2(9): 678-689.
41. Duarte S., et al., (2013) Preventive Cancer Stem Cell‐Based Vaccination Reduces Liver Metastasis Development in a Rat Colon Carcinoma Syngeneic Model. Stem Cells, 31(3): 423-432.
42. Gazdar A F., et al., (1980) Continuous, clonal, insulin- and somatostatin-secreting cell lines established from a transplantable rat islet cell tumor. Proceedings of the National Academy of Sciences of the United States of America, 77(6): 3519-3523.
43. Sharma N K., et al., (1999) A Novel Immunological Model for the Study of Prostate Cancer. Cancer Research, 59(10): 2271-2276.

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