CN112877395A

CN112877395A - Jade in-vitro efficacy evaluation method

Info

Publication number: CN112877395A
Application number: CN202110188442.3A
Authority: CN
Inventors: 杜杰; 高香云; 王征; 林春轶
Original assignee: Beijing Saifu Pharmaceutical Research Institute Co ltd
Current assignee: Beijing Saifu Pharmaceutical Research Institute Co ltd
Priority date: 2021-02-19
Filing date: 2021-02-19
Publication date: 2021-06-01

Abstract

The invention provides a jade in-vitro efficacy evaluation method, which comprises the following steps: providing a transwell chamber comprising an upper chamber and a lower chamber; adding cells and a culture medium into the lower chamber, adding a solution containing jade into the upper chamber, and standing for drug incubation; and taking out the upper chamber, detecting the cell activity in the lower chamber, and calculating the cell activity rate. The method utilizes a transwell chamber to perform cell and drug co-incubation, active ingredients in the jade extract can act on lower chamber cells to inhibit or promote cell proliferation, and other impurities do not influence the judgment of test results. The method has important significance for in vitro pharmacodynamic research of the jade and has good application prospect.

Description

Jade in-vitro efficacy evaluation method

Technical Field

The invention belongs to the field of medicines, and particularly relates to a jade in-vitro efficacy evaluation method.

Background

For a long time, cell-based in vitro assays for drug activity have been essential in pharmacological research, and in particular for drug screening, analysis of the mechanism of action is an essential technique.

When the effect of the drug on cells is detected, the drug is generally directly contacted with the cells after the cells are inoculated on a cell culture plate, and the activity of the cells is detected by utilizing MTT and CCK-8, so that the drug activity detection method is used for detecting the activity of the drug which is easily dissolved in water or can be dissolved in water by adding a certain cosolvent. The jade components to be detected are complex, and extract is difficult to obtain, and the general detection method causes direct contact of particles with cells, so that on one hand, the state of the cells is influenced, and on the other hand, the particles are adsorbed on the bottom of the plate and are difficult to clean, so that the detection result is influenced.

It is noted that the information disclosed in the foregoing background section is only for enhancement of background understanding of the invention and therefore it may contain information that does not constitute prior art that is already known to a person of ordinary skill in the art.

Disclosure of Invention

The invention mainly aims to overcome at least one defect in the prior art and provide a jade in-vitro efficacy evaluation method to solve the problem that the existing method is difficult to effectively evaluate the jade in-vitro efficacy.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides a jade in-vitro efficacy evaluation method, which comprises the following steps: providing a transwell chamber comprising an upper chamber and a lower chamber; adding cells and a culture medium into the lower chamber, adding a solution containing jade into the upper chamber, and standing for drug incubation; and taking out the upper chamber, detecting the cell activity in the lower chamber, and calculating the cell activity rate.

According to one embodiment of the invention, the solution containing jade is allowed to stand and then effective components are leached out and act on cells in the lower chamber, wherein the effective components comprise one or more of trace elements such as selenium and strontium.

According to one embodiment of the invention, the bottom of the upper chamber is an exchange membrane, which is a Polycarbonate (PET) membrane. According to one embodiment of the invention, the pore size of the membrane is between 0.4 μm and 8 μm, preferably 0.4 μm.

According to one embodiment of the invention, the cell concentration in the lower chamber is 2.5X 10⁴～1.25×10⁵cell/mL, cell plating volume of 400 u L.

According to one embodiment of the invention, the jade is present in the upper chamber at a concentration of 1mg/mL to 120 mg/mL.

According to one embodiment of the invention, the upper chamber is removed after the drug incubation for 24-72 hours at rest.

According to one embodiment of the invention, after the upper chamber is taken out, the culture medium in the lower chamber is discarded and cleaned, and the fresh culture medium and the detection reagent are added into the lower chamber, and the cell activity is detected after continuous culture for 3-5 h.

According to one embodiment of the invention, the detection reagent is selected from one or more of CCK-8, MTT.

According to one embodiment of the invention, the cells are selected from one or more of human neuroblastoma cells, human lymphoma cells, melanoma cells, human normal thyroid cells and human small airway epithelial cells.

According to the technical scheme, the invention has the beneficial effects that:

the invention provides a novel jade in-vitro evaluation method, which comprises the steps of culturing through a Transwell chamber, incubating jade medicines and cells together, and then evaluating the activity of the medicines by detecting the cell viability. By using the method of the invention, the effective components in the jade can penetrate through the membrane of the upper chamber of the Transwell to act on the cells of the lower chamber, so that the cell proliferation is inhibited or promoted, and other impurities do not influence the judgment of the test result. The method has important significance for in vitro pharmacodynamic research of the jade and has good application prospect.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention.

FIG. 1 is a schematic flow chart of a method for evaluating the in vitro efficacy of a jade according to an embodiment of the present invention;

FIG. 2 is a schematic structural diagram of in vitro efficacy evaluation of jade using a transwell chamber according to one embodiment of the present invention;

fig. 3 to 5 show inverted microscope images of cells after incubation for 72h in the different treatment regimes of comparative example 1, respectively.

Wherein the reference numerals are as follows:

100: upper chamber

101: exchange membrane

200: lower chamber

300: cells

400: culture medium

500: jade-containing solution

Detailed Description

The following presents various embodiments or examples in order to enable those skilled in the art to practice the invention with reference to the description herein. These are, of course, merely examples and are not intended to limit the invention. The endpoints of the ranges and any values disclosed in the present application are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to yield one or more new ranges of values, which ranges of values should be considered as specifically disclosed herein.

FIG. 1 is a schematic flow chart of a method for evaluating the in vitro efficacy of a jade according to an embodiment of the present invention; FIG. 2 shows a schematic structure diagram of the evaluation of the efficacy of jade in vitro using a transwell cell according to one embodiment of the present invention. With reference to fig. 1 and 2, the present invention provides a method for evaluating the in vitro drug effect of jade, comprising: providing a transwell chamber comprising an upper chamber 100 and a lower chamber 200; adding cells 300 and culture medium 400 into lower chamber 200, adding jade-containing solution 500 into upper chamber 200, standing for drug incubation; and taking out the upper chamber 100, detecting the activity of the cells in the lower chamber 200, and calculating the cell activity rate.

According to the invention, at present, more and more researches show that the jade not only has a health-care effect, but also has a certain pharmaceutical effect, and an effective detection method is provided to research the action mechanism of the jade, which is a problem to be solved in the field. When the effect of the drug on the cells is detected, the drug is generally directly contacted with the cells after the cells are inoculated on a cell culture plate, and then the activity of the cells is detected by utilizing MTT and CCK-8. However, because the jade components to be detected are complex and difficult to extract to obtain extract, the general detection method causes the particles to directly contact with the cells, on one hand, the state of the cells is influenced, on the other hand, the particles are adsorbed on the bottom of the plate and are difficult to clean, and the detection result is influenced. Therefore, the inventor of the invention finds that the in-vitro medicinal activity of the jade can be effectively evaluated by constructing a jade in-vitro medicinal effect evaluation model by using a transwell chamber.

The method for evaluating the in vitro efficacy of jade according to an embodiment of the present invention will be described in detail below with reference to fig. 1 and 2.

First, a transwell chamber is provided. As shown in FIG. 2, the transwell cell comprises an upper chamber 100 and a lower chamber 200, wherein the bottom of the upper chamber 100 is an exchange membrane 101, the exchange membrane 101 can be made of Polycarbonate (PET) membrane, and the pore diameter of the exchange membrane is 0.4 μm to 8 μm, preferably 0.4 μm.

Next, the cells are cultured, and the cultured cells 300 are placed in the lower chamber 200 containing the culture medium 400. Preferably, the cell concentration in lower chamber 200 is 2.5X 10⁴～1.25×10⁵cell/mL, cell plating volume of 400 u L. The medium may be a cell culture medium commonly used in the art, for example, a DMEM medium, a MEM medium, an RPMI-1640 medium, etc., and the present invention is not limited thereto.

Further, a solution 500 containing jade is added to the upper chamber 200, and left to stand for drug incubation. Preferably, the jade is ground into 200 mesh jade powder, and then dissolved in the corresponding culture medium for cell culture to obtain the above-mentioned jade-containing solution 500.

According to the invention, jade is a natural ore with more complex components, and the main components of the jade comprise one or more of trace elements such as selenium and strontium. According to the invention, jade drug action detection is combined with transwell cell technology, and a jade-containing solution 500 corresponding to the cell concentration of the lower chamber 200 is prepared and added into the upper chamber 100 for co-incubation, so that the drug active ingredients after jade leaching can enter the lower chamber 200 through the exchange membrane 101 of the upper chamber 100, and thus, the drug active ingredients can play a role in the cells 300. Meanwhile, due to the limitation of the aperture of the exchange membrane 101, larger particle insoluble matters in the upper chamber 100 can be intercepted, so that the influence of the particles on the cell state or the influence of the particles adsorbed at the bottom of the upper chamber on the result can be avoided.

Specifically, the concentration of the jade in the upper chamber is generally 1mg/mL to 120mg/mL, for example, 1mg/mL, 5mg/mL, 10mg/mL, 50mg/mL, 100mg/mL, 120mg/mL, etc. When different cells and different cell concentrations are used, the preferred concentration ranges of the jade also vary. For example, when the cells selected are human neuroblastoma cells, the cell concentration is 1.25X 10⁵When the cell/mL is adopted, the final action concentration of the jade is preferably controlled to be 25 mg/mL-116.7 mg/mL. For another example, the melanoma cell concentration is 2.5X 10⁴controlling the final action concentration of the jade to be 100 mg/mL-120 mg/mL; the concentration of human normal thyroid cell is 1.25 × 10⁵controlling the final action concentration of the jade to be 4 mg/mL-116.7 mg/mL; the concentration of human small airway epithelial cells is 1.25 multiplied by 10⁵cell/mL, and controlling the final action concentration of the jade to be 4-50 mg/mL and the like.

In some embodiments, the time for drug incubation at rest is 24h to 72h, such as 24h, 48h, 50h, 52h, 60h, 65h, 70h, 72h, and the like. The incubation time of the medicine is not too short, otherwise, effective leaching of jade components cannot be realized, and is not too long, otherwise, the influence of the medicine on cells cannot be accurately judged, and the preferable time is 72 h.

Finally, the upper chamber 100 is removed, and the cell viability in the lower chamber 200 is measured to calculate the cell viability rate.

Specifically, the upper chamber 100 is generally removed before the drug incubation is completed, the original culture medium in the lower chamber 200 is discarded, the medium is washed with PBS buffer, and then fresh culture medium and detection reagent are added to the lower chamber 200, and the incubation is continued for 4 hours. After the culture is finished, detecting the activity of the cells, and calculating the cell activity rate. The cell viability rate is calculated as follows:

the aforementioned detection reagent may be selected from one or more of CCK-8 and MTT, and those skilled in the art may select the corresponding detection reagent according to the actual situation, and the present invention is not limited thereto. The cells for detection may be any one or more cells that can be used in vitro culture, such as human neuroblastoma cells, human lymphoma cells, and the like, and the present invention is not limited thereto.

In conclusion, the invention provides a novel jade in-vitro evaluation method, which comprises the steps of culturing through a Transwell chamber, incubating jade medicines and cells together, and then evaluating the medicine activity through detecting the cell activity. By using the method of the invention, the effective components in the jade can penetrate through the membrane of the upper chamber of the Transwell to act on the cells of the lower chamber, so that the cell proliferation is inhibited or promoted, and other impurities do not influence the judgment of the test result. The method has important significance for in vitro pharmacodynamic study of Chinese medicinal extract, especially jade extract, and has good application prospect.

The invention will be further illustrated by the following examples, but is not to be construed as being limited thereto.

The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Cell: human neuroblastoma SH-SY5Y cell (cat # GDC210) purchased from China center for type culture Collection;

reagent: MEM medium (cat # 41500), 0.25% trypsin-EDTA digest (cat # T1300), streptomycin mixed solution (100X) (cat # P1400), CCK-8 kit for cell proliferation and toxicity detection (cat # CA1210) were purchased from Solebao; fetal bovine serum (cat # 22011-; cisplatin injection (6mL:30mg) was purchased from Jiangsu Hawson pharmaceutical industry group, Inc. Jade powder to be measured: purchased from jilin province, pannage development ltd.

The instrument comprises the following steps: an electric heating constant temperature water bath (model: LUX-12) purchased from Beijing Luxi technology Ltd; clean bench (model: DL-CJ-2NDI) available from Haer instruments, Inc., Tokyo, N.K.; inverted microscope (model: DMIL LED) available from Leica; a centrifuge (model: L3-5K) available from available instruments and Equipment Co., Ltd, Hunan; carbon dioxide incubator (model: HF151) available from hong Kong Likang; a multifunctional microplate reader (model: Fluostar Omega with ABS) was purchased from BMG, Germany.

Example 1

1) Cell culture (with recovery, passage)

Cell recovery process: taking out human neuroblastoma SH-SY5Y cell cryopreservation tube from a liquid nitrogen tank, rapidly placing in a 37 ℃ water bath pot, slightly shaking to completely melt, taking out, sucking cell suspension in a centrifuge tube, adding 2mL of culture solution, 1000rpm/min, centrifuging for 5min, removing supernatant, adding 2mL of MEM complete culture medium, uniformly blowing with a suction tube, transferring to a culture bottle, supplementing the culture medium, placing in 37 ℃ and 5% CO₂Culturing in an incubator, observing every other day, and observing whether the cells need to be continuously cultured or need to be subjected to liquid change or subculture.

And (3) cell passage process: after cells are recovered, when adherent cells grow to be about 80% -90% of the bottom of a bottle, the cells need to be subjected to subculture, culture solution in the bottle is sucked out, the cells are washed twice by sterile PBS, residual culture medium is removed, a proper amount of pancreatin is added, the cells are lightly shaken to be uniformly covered, the cells are placed in an incubator, the edge of the cells can be observed to be rounded under a mirror after 2min generally, the cells continuously fall off from the bottom of the bottle after the cells are lightly shaken, complete culture solution with 2 times of volume of the pancreatin is added to stop digestion, the cells are transferred to a centrifuge tube after being uniformly blown, the centrifuge is carried out at 1000rpm/min, supernatant is removed after 5min centrifugation, 2mL of culture solution is added, and the cells are subjected to subculture at a ratio.

2) Cell plating and drug incubation

Taking human neuroblastoma SH-SY5Y cells in logarithmic growth phase, digesting by using 0.25% trypsin, centrifuging at 1000rpm for 5min, and counting the cells;

cell concentration was adjusted to 1.25X 10 with MEM complete medium⁵cell/mL；

To the lower chamber of each Transwell cell was added 400. mu.L of 5X 10⁴Cell suspension, culturing overnight;

taking out 24-well plate with small chamber from 37 deg.C the next day, and directly adding 200 μ L of contrast cisplatin low-medium dosage group into lower chamber at concentrations of 12 μ M, 60 μ M, and 300 μ M, respectively, to obtain final action concentrations of 4 μ M, 20 μ M, and 100 μ M; 200 mu L of the low-medium dosage group of the jade sample to be tested is gently added into each upper chamber, the concentration is 14mg/mL, 70mg/mL and 350mg/mL respectively, the final action concentration is 4.7mg/mL, 23.3mg/mL and 116.7mg/mL respectively, and bubbles are avoided. In addition, a control was set up, i.e. 200 μ LMEM complete medium was added directly to the lower chamber without adding drug to the upper chamber.

The 24-well plate was placed at 37 ℃ and 5% CO₂And (5) standing in an incubator for drug incubation for 72h, and determining a detection time point according to a pre-test result.

3) Cell viability assay

4h before the drug incubation is finished, taking out the upper chamber of the small chamber, discarding the culture medium of the lower chamber, washing for 1 time by PBS, adding 270 mu LMEM culture medium into each hole of the lower chamber, adding 30 mu L CCK-8 detection reagent, putting the lower chamber into an incubator, continuously culturing for 4h, taking out a 24-hole plate, absorbing 100 mu L of solution to be detected by each hole, transferring the solution to a 96-hole enzyme label plate, reading an absorption value by an enzyme label instrument at the wavelength of 450nm, and calculating the cell activity:

TABLE 1 cell viability assay OD values

As can be seen from Table 1 above, the cell viability rates of the cisplatin low dose group (4. mu.M), the cisplatin middle dose group (20. mu.M) and the cisplatin high dose group (100. mu.M) were (31.15. + -. 4.77%), (25.49. + -. 0.35)% and (26.08. + -. 1.55)%; the cell viability rates of the jade low dose group (4.7mg/mL), the middle dose group (23.3mg/mL) and the high dose group (116.7mg/mL) were (95.28 + -2.88)%, (93.27 + -3.02)%, and (82.18 + -6.22)%, respectively. Compared with a control group, the high, medium and low doses of cisplatin and the high dose of jade all have obvious inhibition effect on the in vitro growth of human neuroblastoma cells SH-SY5Y (P is less than 0.001 or P is less than 0.05); the jade medium and low dose groups have no obvious inhibition effect (P is more than 0.05). Therefore, by adopting the method, the effective components in the jade can penetrate through the cell under the action of the membrane of the upper chamber of the Transwell to inhibit cell proliferation, and impurities do not influence the judgment of the test result, so that the in vitro efficacy evaluation of the jade can be effectively carried out.

Comparative example 1

Adding the jade suspension with high concentration of 116.7mg/mL in example 1 into culture well with small chamber or directly adding the jade suspension with low and medium concentration of 4.7mg/mL and 23.3mg/mL into 400 μ L after overnight culture in step 2) in example 1⁴In cell suspension, and the drug cells were incubated for 72 h.

Under-lens observation is carried out by an inverted microscope (10X objective), and fig. 3 to 5 respectively show inverted microscope images of cells incubated for 72h in different treatment modes, wherein fig. 3 is a jade high dose group (using transwell chamber), fig. 4 is a jade low dose group (direct effect of jade), and fig. 5 is a jade medium dose group (direct effect of jade). The results show that the method using the transwell chamber is not interfered by jade impurities, and the morphology of the cells can be clearly seen. However, the experimental hole directly added with the jade suspension has unknown cell morphology, and can not effectively remove the interference of impurity particles directly influencing the fine activity rate and the detection of the absorption value of the experimental end point. The method provided by the invention can enable the effective components to penetrate through the membrane of the upper chamber of the Transwell to act on the cells of the lower chamber, and impurities do not influence the judgment of the test result, so that the method is beneficial to the evaluation of the external drug effect of the jade.

It should be noted by those skilled in the art that the described embodiments of the present invention are merely exemplary and that various other substitutions, alterations, and modifications may be made within the scope of the present invention. Accordingly, the present invention is not limited to the above-described embodiments, but is only limited by the claims.

Claims

1. A jade in-vitro efficacy evaluation method is characterized by comprising the following steps:

providing a transwell chamber comprising an upper chamber and a lower chamber;

adding cells and a culture medium into the lower chamber, adding a solution containing jade into the upper chamber, and standing for drug incubation; and

and taking out the upper chamber, detecting the cell activity in the lower chamber, and calculating the cell activity rate.

2. The method for evaluating the in vitro pharmacological effect of jade according to claim 1, wherein the solution containing jade is allowed to stand and then effective components are leached out and act on cells in the lower chamber.

3. The jade in-vitro efficacy evaluation method according to claim 1, wherein the bottom of the upper chamber is an exchange membrane, and the exchange membrane is a polycarbonate membrane.

4. The method for evaluating the in vitro drug effect of jade according to claim 3, wherein the pore size of the exchange membrane is 0.4 μm to 8 μm.

5. The method for evaluating the in vitro pharmacological effect of jade according to claim 1, wherein said cell concentration in said lower chamber is 2.5 x 10⁴～1.25×10⁵cell/mL, with a cell plating volume of 400. mu.L.

6. The method for evaluating the in vitro efficacy of jade according to claim 1, wherein the concentration of jade in said upper chamber is 1mg/mL to 120 mg/mL.

7. The method for evaluating the in vitro drug effect of the jade according to claim 1, wherein the upper chamber is taken out after the medicine incubation for 24-72 h after the standing.

8. The method for evaluating the in vitro drug effect of the jade according to claim 1, wherein the culture medium in the lower chamber is discarded and cleaned after the upper chamber is taken out, a fresh culture medium and a detection reagent are added into the lower chamber, and the cell activity is detected after the culture is continued for 3-5 h.

9. The method for evaluating the in vitro drug effect of jade according to claim 8, wherein said detection reagent is selected from one or more of CCK-8 and MTT.

10. The method for evaluating the pharmacological effect of a jade stone of claim 1 wherein said cells are selected from one or more of human neuroblastoma cells, human lymphoma cells, melanoma cells, human normal thyroid cells and human small airway epithelial cells.