阅读说明：本技术 芜菁中性多糖在制备肺癌a549细胞调控的药物中的应用 (Application of turnip neutral polysaccharide in preparation of lung cancer A549 cell regulation and control medicine ) 是由 海力茜·陶尔大洪 马晓丽 周凯 陈春丽 丁海燕 杨飞 乔丽洁 李欢欢 李亚童 米合 于 2021-02-04 设计创作，主要内容包括：本发明涉及芜菁中性多糖应用技术领域,具体公开了芜菁中性多糖在制备肺癌A549细胞调控的药物中的应用,所述芜菁中性多糖作用于肺癌A549细胞时,能够有效抑制人肺腺癌A549细胞增殖,促进人肺腺癌A549细胞中Caspase-3蛋白表达量增加。本发明中的芜菁中性多糖能够促进肺癌A549细胞凋亡,从而为治疗肺癌提供理论基础。(The invention relates to the technical field of turnip neutral polysaccharide application, and particularly discloses application of turnip neutral polysaccharide in preparation of a medicament for regulating and controlling lung cancer A549 cells, wherein when the turnip neutral polysaccharide acts on the lung cancer A549 cells, the turnip neutral polysaccharide can effectively inhibit the proliferation of the lung adenocarcinoma A549 cells and promote the increase of Caspase-3 protein expression in the lung adenocarcinoma A549 cells. The turnip neutral polysaccharide can promote apoptosis of lung cancer A549 cells, thereby providing a theoretical basis for treating lung cancer.)

1. Application of turnip neutral polysaccharide in preparing a medicament for regulating and controlling lung cancer A549 cells.

2. The use of the turnip neutral polysaccharide in the preparation of a medicament for regulating lung cancer A549 cells according to claim 1, wherein the turnip neutral polysaccharide is used for preparing a medicament for promoting apoptosis of lung cancer A549 cells.

3. The use of the turnip neutral polysaccharide in the preparation of a medicament for regulating lung cancer A549 cells according to claim 2, wherein the turnip neutral polysaccharide is used for preparing a medicament for regulating Caspase-3 protein expression in human lung adenocarcinoma A549 cells.

4. The use of the turnip neutral polysaccharide in the preparation of a medicament for regulating lung cancer A549 cells as claimed in claim 3, wherein the turnip neutral polysaccharide is used for preparing a Caspase-3 protein expression promoter in human lung adenocarcinoma A549 cells.

5. The use of turnip neutral polysaccharide in the preparation of a lung cancer A549 cell regulation medicament as claimed in claim 4, wherein the administration dose of turnip neutral polysaccharide is 200 μ g/mL of lung cancer A549 cell sap, and the cell density of lung cancer A549 cell sap is 10⁶each.mL^-1。

6. The use of turnip neutral polysaccharide in the preparation of a lung cancer A549 cell regulation medicament according to claim 5, wherein the administration dose of turnip neutral polysaccharide is 400-600 μ g per mL of lung cancer A549 cell sap.

7. The use of the turnip neutral polysaccharide in the preparation of a medicament for regulating lung cancer A549 cells according to claim 1, wherein the turnip neutral polysaccharide is used for preparing a medicament for inhibiting the proliferation of human lung adenocarcinoma A549 cells.

8. The use of the turnip neutral polysaccharide in the preparation of a lung cancer A549 cell regulation medicament as claimed in claim 7, wherein the administration dose of the turnip neutral polysaccharide is 100 μ g/mL of lung cancer A549 cell sap, and the cell density of the lung cancer A549 cell sap is 10⁶each.mL^-1。

9. The use of the turnip neutral polysaccharide in the preparation of a medicament for regulating lung cancer A549 cells according to claim 8, wherein the administration dose of the turnip neutral polysaccharide is 400-600 μ g per mL of lung cancer A549 cells fluid.

Technical Field

The invention relates to the technical field of turnip neutral polysaccharide application, in particular to application of turnip neutral polysaccharide in preparation of a lung cancer A549 cell regulation and control medicine.

Background

Turnip, a brassica species of Brassica of Brassicaceae, is a plant with homology of medicine and food, and is usually used as a medicine by root tuber and seeds. Li Shizhen has a more detailed description of its efficacy in Ben Cao gang mu, and it is named as "Wu Jing" in Tang Dynasty, which is pungent, bitter, flat, nontoxic, ascending and descending, able to sweat, expel energy, induce diuresis, improve eyesight, and remove toxicity. Turnip is widely distributed in high altitude areas such as Xinjiang, Tibet and the like in China. Xinjiang people refer to the Xinjiang people as Changshou cherry tomatoes, and the Xinjiang people refer to Xinjiang ginseng as the Xinjiang ginseng. It is often used to moisten lung, relieve cough and dyspnea. Mainly comprises the following chemical components: flavones, polysaccharides, alkaloids, etc. At present, the separation and identification of the turnip root tuber show that the turnip has the effects of resisting aging, resisting radiation, relieving cough and asthma, protecting the liver and the like.

Lung cancer is one of the most common malignant tumors, and the incidence rate of lung cancer is high worldwide, and the lung cancer is the disease with the highest incidence rate and mortality rate in tumors. The annual increase in the incidence of lung cancer has become a very serious social problem. Different malignant tumors are controlled by different internal mechanisms, and the apoptosis of tumor cells needs to be promoted from a mechanism level, so that the problem of finding a substance capable of effectively promoting the apoptosis of cancer cells is difficult.

Disclosure of Invention

In order to solve the technical problems, the invention provides application of turnip neutral polysaccharide in preparing a lung cancer A549 cell regulation medicament.

Further, the turnip neutral polysaccharide is used for preparing a medicament for promoting the apoptosis of lung cancer A549 cells.

Further, the turnip neutral polysaccharide is used for preparing a medicament for regulating and controlling the expression of Caspase-3 protein in the human lung adenocarcinoma A549 cells.

Further, the turnip neutral polysaccharide is used for preparing a Caspase-3 protein expression promoter in a human lung adenocarcinoma A549 cell.

Further, the dosage of the turnip neutral polysaccharide is 200-600 mu g per mL of lung cancer A549 cell sap, and the cell density of the lung cancer A549 cell sap is 10⁶each.mL^-1(the 10)⁶Cell densities on the order of magnitude are all acceptable).

Further, the dosage of the turnip neutral polysaccharide is 400-600 mu g of lung cancer A549 cell sap per mL.

Further, the turnip neutral polysaccharide is used for preparing a medicament for inhibiting the proliferation of the human lung adenocarcinoma A549 cells.

Further, the dosage of the turnip neutral polysaccharide is 100-600 mu g-mL of the lung cancer A549 cell sap per mL^-1The cell density of the lung cancer A549 cell sap is 10⁶each.mL^-1(the 10)⁶Cell densities on the order of magnitude are all acceptable).

Further, the dosage of the turnip neutral polysaccharide is 400-600 mu g of lung cancer A549 cell sap per mL.

Compared with the prior art, the invention has the beneficial effects that:

1. the invention provides application of turnip neutral polysaccharide in preparing a medicament for promoting lung cancer A549 cell apoptosis;

2. the turnip neutral polysaccharide can promote the increase of the expression level of Caspase-3 protein in a human lung adenocarcinoma A549 cell;

3. the turnip neutral polysaccharide can promote the increase of the expression level of Caspase-3 protein in a human lung adenocarcinoma A549 cell and promote apoptosis;

4. the turnip neutral polysaccharide can inhibit the proliferation of human lung adenocarcinoma A549 cells, so that the turnip neutral polysaccharide has an inhibiting effect on lung cancer;

5. the turnip neutral polysaccharide can be used as a potential substance in the preparation of medicaments for treating lung cancer.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a graph showing the growth of A549 cells in example 1 of the present invention;

FIG. 2 is a diagram of the morphology of cells under different treatments in example 1 of the present invention;

wherein, FIG. 2A shows a cell morphology without drug intervention;

FIG. 2B shows a cytomorphogram following 10 μ g/mL cisplatin drug intervention;

FIG. 2C shows the cell morphology under 400 μ g/mL turnip neutral polysaccharide drug intervention;

FIG. 2D shows the cell morphology under 100 μ g/mL turnip neutral polysaccharide drug intervention;

Detailed Description

The following detailed description of specific embodiments of the invention is provided, but it should be understood that the scope of the invention is not limited to the specific embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention. The experimental methods described in the examples of the present invention are all conventional methods unless otherwise specified.

Example 1:

example 1 provides an application of turnip neutral polysaccharide in preparing a lung cancer A549 cell-regulated medicament, which comprises the following steps:

materials and instruments

(1) Cell line

A549 lung cancer cell strain 1 is purchased from Wuhan Punuoise Life technologies.

(2) Medicine

Cisplatin (Beijing Solaibao science and technology Co., Ltd.)

The turnip neutral polysaccharide BRNP (self-made in laboratories) has the specific preparation method disclosed in patent CN107011453A, and the alias of turnip is Uighur;

(3) primary reagent

F-12k medium (Wuhan Punuo race Life technologies Co., Ltd., PM150910-500)

0.25% Trypsin (Gibco Corp.)

Fetal bovine serum (Gibco Co.)

Double antibody (Gibco company)

T25 cell culture bottle (corning company)

15. 50mL centrifuge tube (NEST Co.)

60mm cell culture dish (NEST Co., Ltd.)

1000 μ L pipette (eppendorf, Germany)

75% ethanol disinfectant (Hebei kang Ji medicine machinery Co., Ltd.)

0.22 μm sterilizing head (millipore corporation)

Human Caspase-3Elisa kit 96T (Wuhan Elerette Biotech GmbH)

CCK-8 kit 100T (Biosharp company)

(4) Apparatus and device

HERA cell 150 type cell incubator (Thermo company, USA)

Refrigerator (Haier Co.)

Biological safety console (U.S. Thermo company)

Inverted microscope (Motic AE31 Co.)

Centrifuge (sartori mu s company)

NDO-601SD type drying cabinet (Japanese EYELA company)

Second, concrete Experimental method

(1) Cell culture

Taking a bottle of cryopreservation tube out of a refrigerator at minus 80 ℃, quickly thawing in a water bath at 37 ℃ to dissolve, spraying 75% alcohol on the surface of the cryopreservation tube, taking the tube to an operation table, transferring the thawed cell suspension into a 15mL centrifuge tube by using a 1000-L pipette, adding 3mL of complete culture solution into the centrifuge tube, centrifuging (1000 r.min < -1 > and 5min), adding supernatant, adding 2mL of complete culture solution into the centrifuge tube, blowing uniformly, transferring the cell suspension into a T25 cell culture bottle, adding 3mL of complete culture solution, uniformly distributing cells by using a splay mixing method, marking cell varieties and dates, observing morphological characteristics of the cells under a microscope, spraying 75% alcohol on the surface of the culture bottle, and placing the culture bottle into an incubator (37 ℃, 5% CO2) for culture.

(2) Determination of cell growth curves

Human lung cancer A549 cells were digested with 1ml of 0.25% pancreatic enzyme, counted, and cell density was adjusted to 5X 10⁴And each well is paved into a 96-well plate by 100 mu L, the first row and the last row are respectively filled with 100 mu L PBS, the rest rows represent one day, after the plates are paved, the 96-well plate is placed into a constant temperature incubator to be cultured for 24h, each well added with the cells is taken out to be added with 10 mu L CCK-8, the constant temperature incubator is incubated for 1h, the OD value is measured at 450nm, the operation is repeated, the OD value is measured for seven consecutive days, six repeated wells are arranged every day, and a cell growth curve is drawn. Data are presented as mean ± standard deviation and all experimental results were repeated 3 times.

(3) CCK-8 method for detecting influence of neutral polysaccharide on human lung cancer A549 cell proliferation activity

The Cell Co micro-typing Kit-8 (CCK-8 for short) reagent can be used for conveniently and accurately analyzing Cell proliferation and toxicity. This reagent contains WST-8 (chemical name: 2- (2-Methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2, 4-disulfonic acid phenyl) -2H-tetrazole monosodium salt) which is reduced by dehydrogenases in cells to a yellow Formazan product (Formazan dye) with high water solubility under the action of the electron carrier 1-Methoxy-5-methylphenazinium dimethyl sulfate (1-Methoxy PMS). The amount of formazan produced was proportional to the number of living cells. Therefore, this property can be used to directly perform cell proliferation analysis.

The cell suspension was prepared by the method described in (1) above, and the cell concentration was adjusted to 5X 10⁴each.mL^-1Inoculating cells into a 96-well plate, wherein each well is 100 mu L, inoculating 3 culture plates, and culturing the cells; after 24h, the medium was aspirated and the dosing was started, 100. mu.L per well.

The experimental groups were as follows: 1. blank group (without cell, with culture medium); 2. negative control group (without drug, only cells and culture medium added); 3. turnip neutral polysaccharide group (100, 200, 400, 600. mu.g. mL)^-1Turnip neutral polysaccharide); 4. cis-platinum group (5, 10, 20. mu.g. mL)^-1) And filling each of 6 multiple wells with PBS, placing the wells in an incubator for continuous culture after adding drugs, taking out the culture plate after 24 hours, adding 10 mu L of CCK-8 solution, culturing for 1 hour, measuring the OD value at 450nm, and calculating the inhibition rate. Data are presented as mean ± standard deviation and all experimental results were repeated 3 times.

The inhibition rate is 1- (experimental group-blank group)/(control group-blank group) × 100%

(4) Morphological observation of cells

Inoculating human lung adenocarcinoma A549 cells in logarithmic growth phase to a 96-well plate, and performing cell culture; and (4) after 24h, removing the culture solution, adding turnip neutral polysaccharide with different concentrations, respectively incubating for 24h, observing the morphological change of cells under a microscope, photographing, and recording the result.

(5) Enzyme-linked immunosorbent assay for detecting Caspase-3 protein expression in human lung adenocarcinoma A549 cells

(5.1) cell culture: the cell culture was carried out according to the procedure in (1).

(5.2) administration of cells

Human lung adenocarcinoma A549 cells were digested with 1ml of 0.25% pancreatic enzyme, counted, and the cell density was adjusted to 1X 10⁶each.mL^-11mL per well was plated in 6-well plates, and the 6-well plates were placed in a constant temperature incubator for 24 hours, followed by group administration:

grouping e.g. ofThe following: 1. negative control group (without drug, only cells and culture medium added); 2. positive control group (cisplatin 10. mu.g. mL)^-1)3, turnip neutral polysaccharide group (including low dose group 200. mu.g. mL)^-1Medium dose group 400. mu.g.mL^-1High dose group 600. mu.g/mL^-1Turnip neutral polysaccharide). The administration is carried out for 24h and then the subsequent treatment is carried out.

(5.3) preparation of specimens

The cells to be administered for 24h were removed from the incubator, washed 3 times with pre-cooled PBS, digested with pancreatin, and centrifuged (1000 r. min)^-15min), discarding the supernatant, washing the collected cells with precooled PBS 3 times, adding 200. mu.L PBS to the cells for resuspension, repeatedly freezing and thawing the cells 3 times, and centrifuging the extract (1500 r. min.)^-110min), taking the supernatant for detection.

(5.4) operating procedure

The enzyme-linked immunosorbent assay (ELISA) method is adopted to measure the expression of Caspase-3 protein according to the specific steps provided by the kit. And (3) measuring the absorbance value by using a microplate reader, and calculating Caspase-3 protein expression through a standard curve. The specific operation is as follows:

respectively setting a standard hole, a blank hole and a sample hole, adding 100 mu L of standard substance diluted by times into the standard hole, adding 100 mu L of standard substance and sample diluent into the blank hole, adding 100 mu L of sample to be detected into the rest holes, setting 3 multiple holes in each group, covering a film on an ELISA plate, and incubating for 90 minutes at 37 ℃; the liquid in the holes is thrown away completely without washing. Adding 100 mu L of biotinylated antibody working solution into each hole, adding a film on an ELISA plate, and incubating for 1 hour at 37 ℃; throwing off the liquid in the holes, adding 200 mu L of washing liquid into each hole, soaking for 1.5 minutes, absorbing the liquid in the ELISA plate, repeating the plate washing step for 3 times, immediately carrying out the next operation after the plate washing is finished, and not drying the micropore plate; adding 100 mu L of the working solution of the enzyme conjugate into each well, adding a covering film, and incubating for 30 minutes at 37 ℃; discarding liquid in the holes, spin-drying, and washing the plate for 5 times; adding 90 mu L of substrate solution into each hole, adding a film on an enzyme label plate, and incubating for about 15 minutes at 37 ℃ in a dark place; adding 50 mu L of stop solution into each hole to stop the reaction; (the order of addition of the stop solution should be as identical as possible to that of the substrate solution) the optical density of each well was measured at a wavelength of 450nm using a microplate reader.

And after measuring the optical density OD value, calculating the OD value of each group of multiple wells, and subtracting the OD value of the blank well from the average OD value of each standard substance to obtain a correction value. And (4) drawing a standard curve of four-parameter logic on logarithmic coordinate paper by taking the concentration as an abscissa and the OD value as an ordinate, and removing the value of a blank group during drawing. And obtaining the concentration of the sample according to the drawn standard curve and the measured OD value of the sample.

Third, statistical analysis

The data analysis and the drawing are carried out by software such as SPSS25, Origin2015 and the like, and the single-factor analysis of variance is adopted among a plurality of groups, wherein the difference of the data is shown when P is less than 0.05, and the significant difference of the data is shown when P is less than 0.01.

Fourth, experimental results

(1) Cell growth curve

As can be seen from fig. 1: the OD value shows a growing trend in five days before cell culture, particularly reaches the highest peak in the fifth day, and the larger the OD value is, the more the number of living cells is, the OD value is reduced at the beginning of the sixth day, and the number of living cells is reduced. The data analysis shows that: there was a significant difference between day one and day two (P <0.01), and it was considered that day two a549 cells were grown to logarithmic growth phase, so that two days later were passaged once, and the subsequent cells could be cultured for the same time before drug intervention.

(2) Effect of cisplatin on human Lung adenocarcinoma A549 cell proliferation

TABLE 1 Effect of cisplatin on human Lung adenocarcinoma A549 cell proliferation (x + -s, n ═ 6)

Note: and a, compared with a negative control group, P is less than 0.01, the administration lasts 24 hours, and the cisplatin has significant difference between the administration groups with low concentration of 5 mu g/mL, medium concentration of 10 mu g/mL and high concentration of 20 mu g/mL.

As can be seen from Table 1, the inhibition effect of cisplatin on human lung adenocarcinoma A549 cells is enhanced and concentration-dependent increase is shown along with the increase of the administration dose of the control medicament at the same administration time. The cisplatin low concentration 5 mug/mL and the cisplatin medium concentration 10 mug/mL and the cisplatin high concentration 20 mug/mL have significant difference, but the cisplatin medium concentration and the cisplatin high concentration have no significant difference, and the IC50 is close to the cisplatin medium concentration group, so the cisplatin subsequent administration adopts 10 mug/mL.

(3) Effect of turnip neutral polysaccharide on human Lung adenocarcinoma A549 cell proliferation

TABLE 2 Effect of turnip neutral polysaccharide on human Lung adenocarcinoma A549 cell proliferation (x + -s, n ═ 6)

Note: a P <0.01 vs and negative control group, and administration for 24h, there was also significant difference between the administration groups.

With the same administration time, the inhibition effect of the polysaccharide on human lung adenocarcinoma A549 cells is enhanced and the concentration dependence is increased along with the increase of the administration dose of the turnip neutral polysaccharide.

(4) Effect of different concentrations of turnip neutral polysaccharide on A549 cell morphology

As shown in FIG. 2, the cells in FIG. 2A are normal in morphology, fusiform and dense in cell growth; FIG. 2B cells were morphologically altered, most of the cells shriveled, and decreased in number; FIG. 2C shows the change of cell morphology, which is crinkle-like and rounded; FIG. 2D shows a change in morphology of a small number of cells, which grew slowly and shriveled into a round shape compared to the negative control.

(5) Caspase-3 protein expression in human lung adenocarcinoma A549 cell

TABLE 3 Caspase-3 protein expression in human lung adenocarcinoma A549 cells (x + -s, n-6)

Note: the administration is carried out for 24h, a P <0.01 vs and a negative control group.

As shown in Table 3, with the same administration time and the increase of the administration dose of turnip neutral polysaccharide (400-600. mu.g/mL), the expression of Caspase-3 protein in the human lung adenocarcinoma A549 cells is obviously increased, and the apoptosis of the cells is promoted, so that the drug toxicity is possibly related to the mechanism for promoting the apoptosis.

In conclusion, the inhibition rate gradually increases with the increase of the administration dose; the form of A549 cells can be changed by different concentrations of turnip neutral polysaccharide; with the same administration time and the increase of the administration dose of the turnip neutral polysaccharide, the expression level of Caspase-3 protein in the human lung adenocarcinoma A549 cells is increased, and the apoptosis is promoted.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.