Method for treating cancer by intratumoral injection of variant collagenase
A technology of collagenase mutation, applied in the direction of antineoplastic drugs, pharmaceutical formulations, medical preparations containing active ingredients, etc., can solve the problem that the application of mutant collagenase has not yet been established, and achieve the effect of reducing stiffness and volume
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Embodiment 1
[0066] Example 1 Injection of variant collagenase based on B16F10 melanoma mouse model
[0067] Instruments and Materials
[0068] (1) The method of constructing the mouse model is to transplant the mouse melanoma cell B16F10 into C57BL / 6 mice subcutaneously, and select 100 mm 3 , 300mm 3 , 600mm 3 and 1000mm 3 The volume of tumor tissue was observed.
[0069] (2) The tumor tissues of mice of different sizes were selected for experiments at different doses of collagenase. The grouping of the variant collagenases at different doses is as follows:
[0070] Dose group A (0.15mg / 100μl): the mouse numbers are 5-N, 5-L, 3-N, and 5-LL; the corresponding tumor tissue volume is 300mm, respectively 3 , 394mm 3 , 467mm 3 and 1139mm 3, the mice numbered 5-N were injected with 100 μl of 0.9% phosphate buffered saline (PBS), and the mice numbered 5-L, 3-N and 5-LL were injected with 0.15 mg / 100 μl of variant collagen, respectively enzymes;
[0071] Dose group B (0.05mg / 200μl): th...
Embodiment 2
[0079] Example 2 Multi-site injection of variant collagenase based on B16F10 melanoma mouse model
[0080] Instruments and Materials
[0081] The mouse model is C57BL / 6 mice transplanted with melanoma B16F10. Different volumes of tumor tissue are selected for multi-point injection (specifically, 4 points of injection, 50 μl per point) for experiments (for specific injection methods, see Figure 7 ). The specific grouping methods and injection doses are as follows:
[0082] Group A: The mice were numbered 3-RR, 4-L and 5-R, and the corresponding tumor tissue volumes were 1055 mm 3 , 449mm 3 and 423mm 3 , the doses of variant collagenase injected into 3-RR, 4-L and 5-R were 0.025 mg / 200 μl, 0.01 mg / 200 μl and 0.005 mg / 200 μl, respectively (for details, see Figure 8 );
[0083] Group B: The mouse numbers are 4-N and 3-R, respectively, and the corresponding tumor tissue volumes are 2690 mm, respectively 3 and 2354mm 3 , the dose of 4-N injection of variant collagenase is ...
Embodiment 3
[0089] Example 3 B16F10 melanoma mouse model injected with variant collagenase
[0090] Instruments and Materials
[0091] B16F10 melanoma cell line, 6-8 week old C57BL / 6 mice.
[0092] H&E staining experimental materials: fixative, hematoxylin staining solution, eosin staining solution, dilute hydrochloric acid ethanol solution, culture flask, petri dish, ophthalmic forceps, cover glass, slide glass, microscope.
[0093] Materials for immunohistochemistry / fluorescence analysis: sections, reagents / kits, instruments / consumables.
[0094] experimental method
[0095] (1) Model construction: The mouse melanoma cell line B16F10 live cells were allografted into C57BL / 6 mice subcutaneously, and volume detection and anatomical analysis of different volumes were performed, and tissue sections were subjected to H&E / collagen staining and immunohistochemical / fluorescence analysis. .
[0096] (2) Gradient administration of variant collagenase and optimization of conditions: After admi...
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