Kit containing trypsin inhibitor and application thereof
A technology of trypsin inhibition and kit, which is applied in the field of enzyme engineering, can solve the problems of low single cell yield and cell viability, and achieve the effect of promoting further research, high yield and high viability extraction
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[0035] In addition, the kit of the present invention can be used to separate single cells from pancreatic tumor samples, thereby facilitating the analysis of pancreatic tumor samples at the cellular level. Therefore, according to a preferred embodiment of the present invention, the pancreatic sample is a pancreatic tumor sample (pancreatic tumor tissue).
[0036] The kit of the present invention can also be used to separate single cells from non-tumor pancreas samples (such as normal pancreas at the site of the tumor), thereby facilitating the analysis of the influence of tumors on the pancreas at the cellular level. Therefore, according to another aspect of the present invention A preferred embodiment, the pancreas sample is the normal pancreas (normal pancreas tissue) where the tumor is located.
[0037] That is, preferably, the pancreatic sample is normal pancreatic tissue and / or pancreatic tumor tissue.
[0038] The present invention will be described in detail below by w...
Embodiment 1
[0045] This example is used to illustrate the method of isolating single cells from normal pancreatic tissue (normal pancreas at the tumor site) using the kit containing trypsin inhibitor of the present invention, wherein the tissue preservation solution, digestion solution, and lysate used are and washing liquid are as shown in Table 1, the enzyme activity unit value and concentration of each component in every liter of digestive juice are as shown in Table 2, and the preparation method of digestive juice is: mix type VIII collagenase, Dispase II and trypsin inhibitor , use a solvent to dissolve the mixed powder, then add DNaseI and mix well.
[0046] The samples were divided into five groups for experimentation (see Table 3), and the specific operation steps were as follows:
[0047] 1) In the tissue preservation solution, use sterile ophthalmic scissors to trim off the fiber, fat and necrotic tissue on the specimen (the normal pancreatic tissue size is about 1.5×1.5×0.5cm ...
Embodiment 2
[0069] This example is used to illustrate the method of isolating single cells from pancreatic-derived tumor tissue using the kit containing trypsin inhibitors of the present invention, wherein the tissue preservation solution, digestion solution, lysate and washing solution used are shown in Table 1 , the enzyme activity unit value and the concentration of each component in every liter of digestive juice (preparation method is the same as embodiment 1) are as shown in I-1 of table 2.
[0070] 1) In the tissue preservation solution, use sterile ophthalmic scissors to trim off the pancreatic tumor sample (1.5×1.5×0.5cm 3 , Obtained by surgical resection, the patient has signed the informed consent) on the fiber, fat and necrotic tissue, etc.
[0071] 2) Wash 3 times with sterile PBS.
[0072] 3) Put the sample into a 5 mL sterile EP tube (eppendorf), and add about 500 μl of digestion solution.
[0073] 4) Use sterile ophthalmic scissors to cut the tissue into debris (approxim...
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