CN102876633B - Combined cell model and manufacturing method and application thereof - Google Patents

Abstract

The invention discloses a combined cell model, which comprises a Caco-2 cell model and a rat primary cultured hepatic cell model, wherein the Caco-2 cell model and the rat primary cultured hepatic cell model are separated by a semipermeable membrane. The invention also discloses a method for manufacturing the combined cell model and a method for screening medicines by using the combined cell model. By the combined cell model, the absorption and metabolic processes of the medicines can be detected simultaneously, accurately and efficiently, the detection efficiency is improved, and the combined cell model has the bright market application value.

Description

A kind of associational cells model and its production and use

Technical field

The present invention relates to a kind of associational cells model.

Background technology

According to estimates, in new drug development, clinical detection finds that 50% drug candidate drug effect is undesirable, and 40% drug candidate exists safety issue, causes developing unsuccessfully early development work waste.If can carry out external assessment to the activity of drug candidate and toxicity in advance, can greatly reduce drug development cost.ADME/Tox pattern, drug metabolism and toxicity detecting pattern, that developed recently gets up, brand-new, be on cell levels, absorb in early days, the novel new drug research pattern of distribution, metabolism, removing and toxicity test, it can carry out external assessment to the activity of drug candidate and toxicity in advance, is used widely abroad in recent years in each large pharmacy corporation.

When drug candidate is carried out to early screening, can select corresponding high-throughput ADME/Tox external model according to the needs of research, medicine is carried out to early stage activity/toxicity screening.

Caco-2 cell model, human colon adenocarcinoma cell, the cell model absorbing for drugs.Sun Minjie etc., " foundation and the checking of Caco-2 cell monolayer model ", Chinese Pharmaceutical Journal the 41st the 18th phase of volume of September in 2006 discloses a kind of preparation method of Caco-2 cell model: Caco-2 cell model is cultivated in T-75 culturing bottle, nutrient solution is MEM/EBSS NEAA(pH7.4, containing foetal calf serum 10%, L-glutaminate 2mmol/L, Hepeps10mmol/L, penicillin, Streptomycin sulphate), at 37 ℃, containing 5%CO ₂in incubator, full humidity is cultivated, and after going down to posterity, cell is seeded in 24 hole Millicell culture plate inserts in 1:3 ratio, and inoculum density is that 1mL contains 8 * 10 ⁴individual, every hole 400 μ L, base side adds nutrient solution 600 μ L nutrient solutions to cultivate 21 days, during change liquid.

Rat Primary Hepatocytes model, for the cell model of studying medicament metabolism.Zhou Xinghui etc., " research and the Function Identification thereof of the primary culture model of rats'liver parenchyma ", Chinese Clinical pharmacology and therapeutics, in February, 2005; 10(7): 743-746, a kind of preparation method of Rat Primary Hepatocytes model is disclosed: with two step perfusion methods, prepare primary hepatocyte, after use RPMI1640, DMEM in high glucose (4.5g/LD-glucose) and low sugar DMEM(1.0g/L, D-Glucose) cultivate primary hepatocyte, experimental result shows, before there is propagation in primary hepatocyte, low sugar DMEM and DMEM in high glucose are on the impact of the growth of cell and functional status without significant difference, and the culture effect of RPMI1640 is starkly lower than first two nutrient solution.And use low sugar DMEM culture hepatocyte more to press close to liver cell physiological status in vivo, be more suitable for the cultivation in primary cultured hepatocyte.

The current absorption of cell model drugs and the metabolic process utilized only limits to using in order of individual cells model, can only investigate respectively absorption and the metabolism of medicine, lose time, compared to drug disposition metabolism, study simultaneously, lack liver sausage this important step that circulates, exist more interfering factors to cause detecting inaccurate.

Summary of the invention

In order to address the above problem, the invention provides a kind of associational cells model.

Associational cells model of the present invention comprises Caco-2 cell model and Rat Primary Hepatocytes model, and described Caco-2 cell model and Rat Primary Hepatocytes model are separated by with semi-permeable membranes.Described semi-permeable membranes is polycarbonate membrane, polyester film or cellulose mixture film, and membrane pore size is 0.4 ~ 8.0 μ m.

Described polyester film is Millicell suspension type culture dish.

Described Caco-2 cell model is cell transmembrane resistance value>=550 Ω cm ²clone.Described Caco-2 cell model is that cell transmembrane resistance value is 550 ~ 800 Ω cm ²clone.

Described associational cells model is to be prepared as follows to form:

A, get Caco-2 cell, recovery, adds in DMEM-20% nutrient solution and cultivates, and by 1:3, goes down to posterity, and cultivates after 2 generations, changes the cultivation that continues to go down to posterity of DMEM-10% nutrient solution into;

B, get and be covered with in the culture dish of mouse tail collagen and 3rd ~ 15 generations prepared by step a generation cell arbitrarily, adjusting cell density is 1.5 * 10 ⁵individual/mL, is inoculated into the culture dish that is covered with mouse tail collagen from AP side, in culture dish BL side, adds DMEM-10% nutrient solution, is placed in 37 ℃, containing 5%CO ₂incubator in cultivate, cultured continuously 17 ~ 25 days, during change nutrient solution, must be loaded with the culture dish of Caco-2 cell model;

C, get Rat Primary Hepatocytes, mix after adding RP-MI RPMI-1640, obtain Rat Primary Hepatocytes suspension;

D, get Rat Primary Hepatocytes suspension prepared by step c, adjusting cell density is 1.5 * 10 ⁵individual/ml, is inoculated on the Tissue Culture Plate that is covered with mouse tail collagen, and every hole adds changes 2ml every day liquid, cultivates 1 ~ 5 day, must be loaded with the Tissue Culture Plate of Rat Primary Hepatocytes model;

E, step b gained is loaded with to the culture dish of Caco-2 cell model, is placed on the Tissue Culture Plate that steps d gained is loaded with Rat Primary Hepatocytes model.

In step b, AP side refers to top, and BL refers to bottom.

Wherein, in step b:

The culture dish that is covered with mouse tail collagen is to be prepared as follows: get Millicell suspension type culture dish, adding concentration is the I type mouse tail collagen of 50 μ g/ml, and application of sample amount is 100 μ l/cm ², uv irradiating 1 hour, puts into 37 ℃, 5%CO ₂in incubator, spend the night, PBS cleans 3 times;

Get the 4th generation cell; Cultured continuously 21 days; Change the mode of nutrient solution and change nutrient solution after inoculating 24h, change every other day liquid the last week, change liquid one week rear every day.

Wherein, in steps d, cultivate 3 days.

The present invention also provides a kind of method of preparing aforementioned associational cells model, and it comprises the steps:

A, get Caco-2 cell, recovery, adds in DMEM-20% nutrient solution and cultivates, and by 1:3, goes down to posterity, and cultivates after 2 generations, changes the cultivation that continues to go down to posterity of DMEM-10% nutrient solution into;

B, get and be covered with in the culture dish of mouse tail collagen and 3rd ~ 15 generations prepared by step a generation cell arbitrarily, adjusting cell density is 1.5 * 10 ⁵individual/mL, is inoculated into the culture dish that is covered with mouse tail collagen from AP side, in culture dish BL side, adds DMEM-10% nutrient solution, is placed in 37 ℃, containing 5%CO ₂incubator in cultivate, cultured continuously 17 ~ 25 days, during change nutrient solution, must be loaded with the culture dish of Caco-2 cell model;

C, get Rat Primary Hepatocytes, mix after adding RPMI RPMI-1640, obtain Rat Primary Hepatocytes suspension;

D, get Rat Primary Hepatocytes suspension prepared by step c, adjusting cell density is 1.5 * 10 ⁵individual/ml, is inoculated on the Tissue Culture Plate that is covered with mouse tail collagen, and every hole adds changes 2ml every day liquid, cultivates 1 ~ 5 day, must be loaded with the Tissue Culture Plate of Rat Primary Hepatocytes model;

E, step b gained is loaded with to the culture dish of Caco-2 cell model, is placed on the Tissue Culture Plate that steps d gained is loaded with Rat Primary Hepatocytes model.

Wherein, in step b:

The culture dish that is covered with mouse tail collagen is to be prepared as follows: get Millicell suspension type culture dish, adding concentration is the I type mouse tail collagen of 50 μ g/ml, and application of sample amount is 100 μ l/cm ², uv irradiating 1 hour, puts into 37 ℃, 5%CO ₂in incubator, spend the night, with PBS, clean 3 times;

Get the 4th generation cell; Cultured continuously 21 days; Change the mode of nutrient solution and change nutrient solution after inoculating 24h, change every other day liquid the last week, change liquid one week rear every day.

Wherein, in steps d, cultivate 3 days.

The present invention also provides the sieve prescription method of aforementioned associational cells model, and it comprises the steps:

1. adopt aforementioned associational cells model, get medicine to be checked, from the one side dispenser of Caco-2 cell model;

2. collect Rat Primary Hepatocytes model one side sample, detect, according to evaluation medicine to be checked.

Caco-2 cell model and Rat Primary Hepatocytes model are used as an associational cells model integral body, except overcoming the defect of prior art, can also make associational cells model simulate more exactly human body medicine absorbs and metabolic process, thereby result of study is more accurate, to high-throughout drug screening important in inhibiting.

Yet, research is found, Caco-2 cell model and the Rat Primary Hepatocytes directly prepared by existing method put together, because culture system is different, the reasons such as restraining effect of emiocytosis thing, between two kinds of cells, can influence each other, cause two kinds of cell models to be difficult to become an organic whole, can not reach the object of detection of drugs absorption simultaneously and metabolism.

The present invention with semi-permeable membranes by Caco-2 cell model and Rat Primary Hepatocytes model combination, and to the culture condition of two kinds of cell models-comprise nutrient solution, the parameters such as incubation time are improved, the nutrient solution of Rat Primary Hepatocytes particularly, use prior art to think and cultivated undesirable RPMI RPMI-1640 for Rat Primary Hepatocytes, in the associational cells model preparing, the mutual restraining effect of nothing between two kinds of cells, two kinds of cell models have formed a stable organic whole, absorption and metabolism for detection of medicine, favorable reproducibility, accuracy is high, detection efficiency is high, single Caco-2 cell model and Rat Primary Hepatocytes model are significantly better than using in order, prepare the material prices such as the nutrient solution of associational cells model and culture dish cheap, production cost is low, there is good potential applicability in clinical practice.

Obviously, according to foregoing of the present invention, according to ordinary skill knowledge and the customary means of this area, not departing under the above-mentioned basic fundamental thought of the present invention prerequisite, can also make modification, replacement or the change of other various ways.

The embodiment of form, is described in further detail foregoing of the present invention again by the following examples.But this should be interpreted as to the scope of the above-mentioned theme of the present invention only limits to following example.All technology realizing based on foregoing of the present invention all belong to scope of the present invention.

Accompanying drawing explanation

The form of Caco-2 cell cultures after 10 days in Fig. 1 polyester film

The form of Caco-2 cell cultures after 21 days in Fig. 2 polyester film

The form of Caco-2 cell cultures after 21 days in Fig. 3 transmission electron microscope observing polyester film

Fig. 4 is the primary hepatocyte form of separation just

Fig. 5 cultivates the primary hepatocyte form of 24 hours

Fig. 6 cultivates the primary hepatocyte form of 48 hours

Fig. 7 cultivates the primary hepatocyte form of 3 days

Fig. 8 primary hepatocyte ALB of 1-7 days, BUN secretes situation

Fig. 9 ADME/Tox test system schematic diagram in parallel

2h cellular form before the administration of Figure 10 model group

2h cellular form after the administration of Figure 11 model group

Figure 12 standard substance 270nm color atlas

Figure 13 blank solvent 270nm color atlas

Figure 14 conjunctive model sample 270nm color atlas

Figure 15 substep model sample 270nm color atlas

Figure 16 baicalin canonical plotting

Figure 17 wogonoside canonical plotting

Figure 18 wogonin canonical plotting

The Absorption And Metabolism mode of Figure 19 baicalin

Embodiment

The foundation of embodiment 1 associational cells model of the present invention

1 experiment material

1.1 cell strain Caco-2 cell strains are purchased from U.S. ATCC (American Type Culture Collection).

1.2 laboratory animal SD rats, SPF level, male, 170g-200g, is provided conformity certification number by Sichuan Provincial Academy of Traditional Chinese Medicine animal center: SCXK(river) 2008-19.

1.3 main agents and medicine DMEM substratum, RPMI 1640 substratum, trypsinase, type Ⅳ collagenase is purchased from Gibco company, HBSS, I type mouse tail collagen, EDTA2Na, HEPES, non-essential amino acid is purchased from Sigma company, foetal calf serum is purchased from PAA company, pHGF, Urogastron is purchased from Peprotech inc company, penicillin and Streptomycin sulphate are purchased from North China pharmaceutical Co. Ltd, all the other reagent are domestic analytical pure, the first perfusate is PBS, HBSS or D-Hank ' s liquid, wherein containing EDTA2Na 0.01% ~ 0.03%, HEPES 1.43 ~ 5.72g/L, pH 7.2 ~ 7.5, Hemoperfusion time 5 ~ 30min, 25 ℃ ~ 38 ℃ of perfusion temperature.The second perfusate is PBS, HBSS or D-Hank ' s liquid, calcium ions 2.5 ~ 10mmol/L wherein, and type Ⅳ collagenase 0.02% ~ 0.15%, HEPES1.43 ~ 5.72g/L, pH 7.2 ~ 7.5, Hemoperfusion time 5 ~ 30min, 25 ℃ ~ 38 ℃ of perfusion temperature.Damping fluid used is HBSS, PBS or D-Hank ' s solution, and pH 7.2 ~ 7.5.

1.4 main equipment and instruments: 6 orifice plates (Costar), Millicell suspension type culture dish (polyester film, aperture 1.0 μ m), 90mm flat-panel filter, Millicell-ERS cross-film resistance instrument, ultrapure water system (Millipore, the U.S.), 3111 water-jacket typ CO2 incubator (Forma, the U.S.), DMLL inverted phase contrast microscope (Leica, Germany), XSZ-H7 binocular biological microscope (Chongqing), the double two-sided Bechtop of SW-CJ-2F (Purifying Equipment Co., Ltd., Suzhou), ALLEGRAX-15R tabletop refrigerated centrifuge (Beckman, the U.S.), MLS-3070 high-pressure sterilizing pot (Sanyo, Japan), YDS-50B-80 liquid nitrogen vessel (the dynamo-electric Trade Co., Ltd. in East Asia, Leshan), CP-225D type precise electronic balance (Sartorius), PB-10 acidometer (Sartorius), FD-ID-50 freeze drier (Beijing rich doctor health), 7020 full-automatic biochemical tester (Hitachis, Japan), XW-80A whirlpool DL instrument (Luxi, extra large Qingpu).

2 experimental techniques

2.1 set up Caco-2 cell model

2.1.1Caco-2 the recovery of cell and going down to posterity

From liquid nitrogen, take out Caco-2 cell, put into immediately 37 ℃ of water-baths.After melting fast, Caco-2 cell is transferred in the centrifuge tube that fills 8ml DMEM-20% (37 ℃) nutrient solution, mixes, centrifugal 5min under 900 speed that turn, so, after repeated centrifugation 3 times, obtains Caco-2 cell suspending liquid.Caco-2 cell suspending liquid is transferred in Tissue Culture Flask, puts into constant incubator and cultivate, second day changes liquid, changes every other day liquid later.When culturing bottle inner cell converges while reaching approximately 80% left and right, the old nutrient solution that inclines, adds the digestion of 0.25% trypsinase-0.02%EDTA solution.Observation of cell metamorphosis under inverted phase contrast microscope, after about 2min, it is large that intercellular substance becomes, cell rounding, stops digestion immediately, outwells Digestive system, add 6ml DMEM-20% nutrient solution, gently cell is blown off at the bottom of bottle and mix, be transferred in centrifuge tube centrifugal 5min under 900 speed that turn, remove supernatant liquor, add 6ml DMEM-20% nutrient solution, mix, by 1:3, go down to posterity.With DMEM-20% nutrient solution, continue to cultivate after two generations, change DMEM-10% nutrient solution into and cultivate.

2.1.2Millicell film is coated with paving I type mouse tail collagen

Millicell suspension type culture dish is put into 6 well culture plates, and adding concentration is the I type mouse tail collagen 100 μ l/cm of 50 μ g/ml ²(452 μ l) uses culture plate uviolizing in Bechtop, after 1 hour, puts into 37 ℃, 5%CO ₂in incubator, spend the night, after taking out, with PBS, clean Millicell film 3 times, standby.

2.1.3Caco-2 the inoculation of cell and cultivation

During experiment, get the cell of recovery after the 4th generation, by going down to posterity under 2.1.1 item, adjusting density is 1.5 * 10 ⁵individual/mL, is seeded on Millicell film.In the AP of Millicell film side, add the cell suspension 2ml mixing, BL side adds DMEM-10% nutrient solution 4ml.Cell is placed in to 37 ℃, containing 5%CO ₂incubator in cultivate, inoculation is changed nutrient solution after 24h.Change every other day liquid the last week, change afterwards liquid, cultured continuously 2l days every day.

2.1.4Caco-2 the checking of cell model

1. inverted phase contrast microscope observation of cell form: every day is observation of cell growing state under inverted phase contrast microscope.

After cell inoculation 2h, start adherently, while growing to the 4th day left and right, converge and reach 80%, on film, can see cellular form.As shown in Figure 1, after growing 10 days, cell boundaries is clear, is irregular polygon.As shown in Figure 2, grow 21 days time, each iuntercellular is inlayed arrangement as paving stone, and non-overlapping copies, is typical individual layer form.

2. transmission electron microscope observing cellular form: as shown in Figure 3, during Growth of Cells to 21 day, the brush border being formed by microvillus is fine and close in good order, and tight connection and desmosome between cell appear at chamber side.

3. the mensuration of cross-film resistance value: the cross-film resistance value of measuring every other day Caco-2 cell with Millicell-ERS cross-film resistance instrument, before each measuring resistance, electrode PBS soaked overnight, with 75% alcohol immersion 30min, then soaks 15min with nutrient solution standby before using.

The cross-film resistance value of Caco-2 cell=(measured value-blank film resistance value) * Millicell membrane area

The TEER value of Caco-2 cell was at first 6 days rapid developments, and after 6 days, TEER value maintains in a stable scope, and in the time of 21 days, Caco-2 cell transmembrane resistance value is greater than 550 Ω cm ².

2.1.5 brief summary

The Caco-2 cell model that aforesaid method is set up is reproducible, by resistance value, measures discovery, and in the time of 21 days, TEER value is greater than 550 Ω cm ².

2.2 set up primary cultured hepatocyte.

2.2.1 the foundation of primary cultured hepatocyte

(1) open abdomen, along hunter's line, open abdominal cavity, fully expose hepatic vein and postcava, by eye scissors careful separation, also respectively place a suture line.At the portal vein place apart from approximately 2 centimetres of hepatic portals, cut off one " V " type osculum, insert soft silicone tube fixing, silicone tube connects constant flow pump perfusion device.

(2) after intubate success, with 40ml/min, pour into the first perfusate and cut off postcava abdomen section immediately.When liver color shoals to khaki color gradually, (about 200ml left and right), changes and fills with the second perfusate, to Glisson's capsule, occurs that the depression of be full of cracks or pressure stops (about 130ml left and right) while being difficult for recovering.

(3) careful separation liver is in culture dish, by the HBSS liquid Rapid Cleaning of 4 ℃ 2 times, then adds RP-MI RPMI-1640, carefully tears Glisson's capsule, softly swings hepatic tissue, allows cell free out, to remaining liver fibrous tissue be main.Draw the rough suspension of liver cell, successively through 100 orders and 200 order cell screen filtrations.

(4) cell suspension is with the centrifugal 3min of 500r/min, the supernatant liquor that inclines, centrifugal three times so repeatedly.Then suck cell conditioned medium liquid, mix after adding RP-MI RPMI-1640, obtain primary hepatocyte suspension.

(5) adjusting density is 1.5 * 10 ⁵individual/ml, is inoculated on 6 orifice plates that are covered with mouse tail collagen, and every hole adds 2ml cell suspension, and changes nutrient solution every day.

2.2.2 the checking of primary cultured hepatocyte

1. by the Trypan Blue method of exclusion, measure Activity of hepatocytes: the trypan blue solution of hepatocyte suspension and 0.4% is mixed at 1: 1, with full-automatic cell analyser, calculate viable cell percentage immediately.

Image shows that alive liver cell is bright, circle, and stereoscopic sensation is stronger, and dead liver cell is dyed by indigo plant, and cytolemma is fuzzy damaged, and after counting, liver cell survival rate is at 70%-93%, and cell yield is 1.8 * 10 ⁸-2.5 * 10 ⁸between.

2. inverted phase contrast microscope observation of cell form: every day is observation of cell situation under inverted phase contrast microscope.

Just separated liver cell is single, and circle is bright, and stereoscopic sensation is strong (Fig. 4).After 1 hour, liver cell starts adherent growth, becomes flat.After 24 hours, liver cell is adherent, and volume increases and development towards periphery, and what have is monokaryon, and what have is double-core (Fig. 5).After 48 hours, most of liver cell stretches out pseudopodium, connects and is netted (Fig. 6) gradually.Cultivate under the liver cell mirror of 3 days and connect in flakes each other as seen, visible cell core is monokaryon or double-core (Fig. 7).

3. hepatocyte function is learned and is detected: cultured continuously liver cell 7 days, change liquid every day, and get cell culture supernatant 1ml, after the centrifugal 5min of 3000r/min, get supernatant, full automatic biochemical apparatus is measured ALB, BUN concentration.

As shown in Figure 8, the secreting function of primary hepatocyte started at the 1st, 2 days to recover, and peak appearred in the secreting function of its ALB at the 3rd day, then decline gradually, and the secretion of BUN was its active peak periods at 3-4 days, also fell into a decline thereafter.

2.2.3 brief summary

The present invention has set up primary cultured hepatocyte.By the detection to primary hepatocyte survival rate, the observation of metamorphosis, and the detection to its secreting function, result shows, this tests separated primary hepatocyte survival rate at 70%-93%, its Morphology meets bibliographical information, and the secretion of ALB peaked at the 3rd day, and the secretion of BUN peaked at the 3rd, 4 days.Therefore, take the 3rd day be administration time.

The foundation of 2.3 conjunctive models

When Caco-2 cell model is set up 21 days, with HBSS, clean after Caco-2 cell 3 times, observation of cell under microscope, select cytolemma complete, arrange closely without coming off and resistance value is greater than 550 Ω cm ²caco-2 cell.With HBSS, clean the liver cell 3 times cultivated 3 days, dead cell is washed away as far as possible, see that cellular form is clear under mirror, connection is island, and majority is double-core.Available Caco-2 cell (Millicell film) is proposed, and putting down gently to plant has on hepatocellular 6 orifice plates, obtains associational cells model of the present invention, as shown in Figure 9.

Through observing, between two kinds of cells, without mutual restraining effect, prove that the present invention has successfully set up the associational cells model of absorption and metabolism.

This associational cells model of embodiment 2 use is investigated Herba Sidae Rhombifoliae soup ADME/Tox feature

Herba Sidae Rhombifoliae soup, is traditional Chinese medicine preparation, cures mainly SHAO YANG syndrome and married woman's typhoid fever.Baicalin (BCN), scutellarin (BCL), wogonoside (WGS), wogonin (WGN) are the effective constituent of Herba Sidae Rhombifoliae soup.

The present embodiment is investigated the accuracy of associational cells model of the present invention by the Absorption And Metabolism of Baicalin of Xiao Chaihu Tang (BCN), wogonoside (WGS) and wogonin (WGN), 2 groups of control groups are set, using Caco-2 cell model as blank group, using Caco-2 cell model and Rat Primary Hepatocytes use in order as substep model group.

According to prior art, baicalin (BCN), wogonoside (WGS) and the Absorption And Metabolism mode of wogonin (WGN) in Caco-2 cell model and Rat Primary Hepatocytes model are: the Absorption And Metabolism pattern of BCN as shown in figure 19, in liver cell, contain UGT enzyme, can be by the BCL(scutellarin in medicine) be converted into BCN, thus increase the amount of BCN; WGS by Caco-2 cell model, absorbed and be converted into WGN after be transported to Rat Primary Hepatocytes model, WGN by Caco-2 cell model, absorbed and cross-docking to Rat Primary Hepatocytes model, WGN is in Rat Primary Hepatocytes model, can be converted into WGS and be transported to opposite side, also can cross-docking to opposite side.

Particularly: 1, blank model group is because lacking liver cell model, in its sample finally obtaining, BCN content should be lower than conjunctive model group and substep model group, WGS content should be lower than conjunctive model group and substep model group, and WGN content should be higher than conjunctive model group and substep model group, 2, substep model group and conjunctive model group are compared: in substep model, Caco-2 cell model absorbs after BCN and BCL completely, be transported to again Rat Primary Hepatocytes model, and in conjunctive model group of the present invention, BCN and BCL that Caco-2 cell model absorbs can be transported to Rat Primary Hepatocytes model immediately, strengthened the concentration difference of Caco-2 cell model both sides BCL, promote BCL rapid transport, and then improving BCN content, in conjunctive model BL side sample of the present invention, the concentration of BCN should be higher than the concentration of BCN in substep Model B L side sample, in substep model, Caco-2 cell model absorbs after WGS and WGN completely, all be converted into WGN, WGN is by the metabolism of Rat Primary Hepatocytes model, form with WGN or WGS is transported to opposite side, in conjunctive model group, the WGS that the metabolism of Rat Primary Hepatocytes model generates can be through being transported to Caco-2 in liver sausage circulation once again, be converted into WGN, cause WGN concentration to improve, in conjunctive model BL side sample of the present invention, the concentration of wogonoside (WGS) should be lower than the concentration of substep Model B L side sample, the concentration of wogonin (WGN) should be higher than the concentration of distributed model BL side sample.

1 experiment material and method

1.1 experiment material

1.1.1 given the test agent Herba Sidae Rhombifoliae soup (XCHT) is provided by Sichuan Provincial Academy of Traditional Chinese Medicine Chemistry for Chinese Traditional Medicine institute, lot number 20071220.

1.1.2 cell strain Caco-2 cell strain is purchased from U.S. ATCC (American Type Culture Collection).

1.1.3 laboratory animal SD rat, SPF level, male, 170-200g, is provided conformity certification number by Sichuan Provincial Academy of Traditional Chinese Medicine animal center: SCXK(river) 2008-19.

1.1.4 main agents and medicine: DMEM substratum, RP-MI 1640 substratum, trypsinase, type Ⅳ collagenase is purchased from Gibco company, HBSS, I type mouse tail collagen, EDTA2Na, HEPES, non-essential amino acid is purchased from Sigma company, foetal calf serum is purchased from PAA company, pHGF, Urogastron is purchased from Peprotech inc company, penicillin and Streptomycin sulphate are purchased from North China pharmaceutical Co. Ltd, BCN standard substance (content >=98%), WGN standard substance (content >=98%) are purchased from Nat'l Pharmaceutical & Biological Products Control Institute, WGS standard substance (content >=98%) are purchased from Wei Keqi bio tech ltd, Sichuan, trifluoroacetic acid aqueous solution is purchased from Wo Kai company, Chromatographic Pure Methanol is purchased from U.S. fisher company, all the other reagent are domestic analytical pure.

1.1.5 main equipment and instrument: 6 orifice plates (Costar), suspension type culture dish (polyester film, aperture 1.0 μ m), 90mm flat-panel filter, Millicell-ERS cross-film resistance instrument, ultrapure water system (Millipore, the U.S.), 3111 water-jacket typ CO2 incubator (Forma, the U.S.), DMLL inverted phase contrast microscope (Leica, Germany), XSZ-H7 binocular biological microscope (Chongqing), the double two-sided Bechtop of SW-CJ-2F (Purifying Equipment Co., Ltd., Suzhou), ALLEGRAX-15R tabletop refrigerated centrifuge (Beckman, the U.S.), MLS-3070 high-pressure sterilizing pot (Sanyo, Japan), YDS-50B-80 liquid nitrogen vessel (the dynamo-electric Trade Co., Ltd. in East Asia, Leshan, Waters high performance liquid chromatograph (Waters highly effective liquid phase chromatographic system 2695-2996), the work of Empower Pro chromatographic data, CP-225D type precise electronic balance (Sartorius), PB-10 acidometer (Sartorius), FD-ID-50 freeze drier (Beijing rich doctor health), 7020 full-automatic biochemical tester (Hitachis, Japan), XW-80A whirlpool DL instrument (Luxi, extra large Qingpu).

1.2 experimental technique

1.2.1 grouping and administration

1. conjunctive model group (associational cells model of the present invention): add 2ml Herba Sidae Rhombifoliae soup liquid in Caco-2 cell AL side, liver cell side (BL side) adds 4mlHBSS liquid, collects liver cell side sample after 2 hours, and-20 ℃ frozen.

2. substep model group: add 2ml Herba Sidae Rhombifoliae soup liquid in Caco-2 model AL side, BL side adds 4mlHBSS liquid, collects BL side through absorbing sample after 2 hours, and absorptions sample is added in liver cell model, acted on after 2 hours, collection sample, and-20 ℃ are frozen.

3. blank group: add 2ml Herba Sidae Rhombifoliae soup liquid in Caco-2 model AL side, BL side adds 4mlHBSS liquid, collects BL side sample after 2 hours, and-20 ℃ frozen.

1.2.2 the preparation of standard solution

Precision takes BCN respectively, and WGS and WGN standard substance are appropriate, and with methyl alcohol, be mixed with concentration and be respectively 10 μ g/ml, 9.6 μ g/ml, the hybrid standard product solution of 10 μ g/ml, as standard reserving solution.

2 condition determinations

Chromatographic column: SymmetryTM Shield R18(5.0 μ m, 4.6mm * 250mm)

Moving phase: acetonitrile-0.01% phosphoric acid carries out gradient elution, in Table 1

Table 1 eluent gradient wash-out table

Column temperature: 30 ℃; Flow velocity: 0.45ml/min; Detect wavelength: λ=270nm

3 methodological studies

3.1 method specificities

Record hybrid standard product, blank sample, sample chromatogram figure and their ultraviolet absorpting spectrum, investigate the specificity of analytical procedure.

3.2 linear relationships are investigated

Get hybrid standard storing solution, with methyl alcohol, be diluted to a series of different concentration, by condition determination, measure, measure peak area integrated value, with peak area Y, concentration X is carried out to linear regression.

3.3 Precision Experiment

Get hybrid standard storing solution, be made into three different concentration, each concentration determination 3 times, does withinday precision and investigates.

3.4 repeated experiment

The hybrid standard product of 5 parts of same concentrations of preparation, after dissolve with methanol, ultrasonic 30min, 0.22 μ m membrane filtration, filtrate, as need testing solution, is respectively got 20 μ l sample introductions, records peak area.Calculate each component content, investigate repeatability.

3.5 rate of recovery experiments

The hybrid standard product of 3 different concns of accurate preparation, dissolve with HBSS liquid, and concentration is respectively 2.5 μ g/ml, 1.25 μ g/ml, 0.625 μ g/ml.Ultrasonic 30min, adds methyl alcohol again after freeze drier freeze-drying.Press content assaying method and measure, calculate recovery rate.

3.6 stability experiment

Get Herba Sidae Rhombifoliae soup metabolite need testing solution, respectively 2.5,5,7.5,10,24 hours, sample introduction 20 μ l, measured peak area integrated value, investigate the stability of sample.

The pre-treatment of 3.7 samples: get 1ml sample, freeze drier freeze-drying, adds 0.3ml methyl alcohol, and vortex instrument mixes, ultrasonic 30min gets supernatant liquor after the centrifugal 10min of 15000r/min, standby with 0.22 μ m membrane filtration.

4 experimental results

After 4.1 model group administrations, liver cell changes

As shown in Figure 10 ~ 11, the administration of substep model group, after 2 hours, has no cellular form and changes under mirror; The administration of conjunctive model group is after 2 hours, and under mirror, intercellular substance increases, and island connection becomes not obvious, liver cell volume-diminished, and dead cell increases.

4.2 method specificities

As shown in Figure 12 ~ 15, under this experiment condition, BCN, WGS and WGN retention time are respectively 13min, 21min and 51min.Each peak resolution is greater than 1.5, and in sample, the ultraviolet absorpting spectrum of BCN, WGS and WGN is consistent with reference substance, and BCN has maximum absorption band at 276nm place, and WGS has maximum absorption at 272nm place, and WGN has maximum absorption at 275nm place.

4.2.1 typical curve

Get standard reserving solution, with methyl alcohol, be diluted to 2.5 μ g/ml, 1.25 μ g/ml, 0.625 μ g/ml, 0.31 μ g/ml.Press condition determination sample introduction and measure, measure peak area integrated value, with area, concentration is carried out to linear regression, as shown in Figure 16 ~ 18 and table 2, R value is all greater than 0.99, and linear relationship is good.

Table 2 typical curve and sensitivity

4.2.2 Precision Experiment

Precision is investigated result and is shown, RSD% is all less than 5%, illustrates that present method precision is good, the results are shown in Table 3.

Table 3 Precision Experiment result

4.2.3 repeated experiment

Repeatability is investigated result and is shown, RSD% is all less than 2%, illustrates that present method repeatability is good, the results are shown in Table 4.

Table 4 repeated experiment result

4.2.4 stability

The demonstration of study on the stability result, RSD% is all less than 3%, illustrates that present method has good stability, and the results are shown in Table 5.

Table 5 stability experiment result

4.2.5 average recovery

The average recovery of each reference substance is less than 3% at 73～110%, RSD%, and the rate of recovery is good.It the results are shown in Table 6.

Table 6 average recovery experimental result

The present embodiment has been set up the method that high performance liquid chromatography separation-diode array detector is measured 3 kinds of component contents in Herba Sidae Rhombifoliae soup metabolite simultaneously.Sample separation degree is higher, and specificity is strong, and the rate of recovery, precision, repeatability, stability and quantitative linearity scope all meet the related request of biology sample detection.

The measurement result that baicalin of the present invention (BCN), wogonoside (WGS) and wogonin (WGN) are described is accurately and reliably.

The assay of 4.3 Herba Sidae Rhombifoliae soup Metabolites

Each composition measurement of Herba Sidae Rhombifoliae soup BL side metabolism sample the results are shown in Table 7:

Table 7 Herba Sidae Rhombifoliae soup Metabolite measurement result

With blank group comparison: * P ﹤ 0.05, * * P ﹤ 0.01; With conjunctive model group comparison: #P ﹤ 0.05, ##P ﹤ 0.01.

As shown in Table 7, in the Herba Sidae Rhombifoliae soup metabolite after conjunctive model Absorption And Metabolism, the content of BCN, WGS and WGN is respectively (2.60 ± 0.11) μ g, (2.03 ± 0.11) μ g, (0.65 ± 0.05) μ g; BCN, WGS, WGN in substep model are respectively (2.37 ± 0.37) μ g, (2.72 ± 0.53) μ g, (0.44 ± 0.09) μ g.

In conjunctive model group and substep model group, BCN and WGS content are all higher than blank group, and WGN content is all lower than blank group, with the equal significance of blank group comparing difference (P<0.01); The BCN of conjunctive model group and WGN content are higher than substep model group (P<0.01), and the WGS content of conjunctive model group is lower than distributed model group (P<0.05).

Experimental result shown in table 7 is consistent with BCN, WGS in prior art and WGN Absorption And Metabolism mode, and conjunctive model of the present invention absorption and the metabolic process of detection of drugs are exactly described.

To sum up, the present invention has successfully set up conjunctive model, and two kinds of cell models in conjunctive model are without mutual inhibition, simultaneously absorption and the metabolic process of detection of drugs, accuracy is high, similar to internal metabolism height, for medicine screens the basis that provides good in earlier stage, shorten the cycle of drug development research, raise the efficiency, reduce costs, avoid risk, there is good market using value.

Claims (12)

1. an associational cells model, is characterized in that: it comprises Caco-2 cell model and Rat Primary Hepatocytes model, and described Caco-2 cell model and Rat Primary Hepatocytes model are separated by with semi-permeable membranes.

2. associational cells model according to claim 1, is characterized in that: described semi-permeable membranes is polycarbonate membrane, polyester film or cellulose mixture film, and membrane pore size is 0.4 ~ 8.0 μ m.

3. associational cells model according to claim 2, is characterized in that: described polyester film is Millicell suspension type culture dish.

4. associational cells model according to claim 1, is characterized in that: described Caco-2 cell model is that cell transmembrane resistance value is more than or equal to 550 Ω cm ²clone.

5. associational cells model according to claim 4, is characterized in that: described Caco-2 cell model is that cell transmembrane resistance value is 550 ~ 800 Ω cm ²clone.

6. associational cells model according to claim 1, is characterized in that: it is to be prepared as follows to form:

A, get Caco-2 cell, recovery, adds in DMEM-20% nutrient solution and cultivates, and by 1:3, goes down to posterity, and cultivates after 2 generations, changes the cultivation that continues to go down to posterity of DMEM-10% nutrient solution into;

B, get and be covered with in the culture dish of mouse tail collagen and 3rd ~ 15 generations prepared by step a generation cell arbitrarily, adjusting cell density is 1.5 * 10 ⁵individual/mL, is inoculated into the culture dish that is covered with mouse tail collagen from AP side, in culture dish BL side, adds DMEM-10% nutrient solution, is placed in 37 ℃, containing 5%CO ₂incubator in cultivate, cultured continuously 17 ~ 25 days, during change nutrient solution, must be loaded with the culture dish of Caco-2 cell model;

C, get Rat Primary Hepatocytes, mix after adding RP-MI RPMI-1640, obtain Rat Primary Hepatocytes suspension;

D, get Rat Primary Hepatocytes suspension prepared by step c, adjusting cell density is 1.5 * 10 ⁵individual/ml, is inoculated on the Tissue Culture Plate that is covered with mouse tail collagen, and every hole adds changes 2ml every day liquid, cultivates 1 ~ 5 day, must be loaded with the Tissue Culture Plate of Rat Primary Hepatocytes model;

E, step b gained is loaded with to the culture dish of Caco-2 cell model, is placed on the Tissue Culture Plate that steps d gained is loaded with Rat Primary Hepatocytes model.

7. associational cells model according to claim 6, is characterized in that: in described step b:

The culture dish that is covered with mouse tail collagen is prepared as follows: get Millicell suspension type culture dish, adding concentration is the I type mouse tail collagen of 50 μ g/ml, and application of sample amount is 100 μ l/cm ², uv irradiating 1 hour, puts into 37 ℃, 5%CO ₂in incubator, spend the night, PBS cleans 3 times;

Get the 4th generation cell; Cultured continuously 21 days; Change the mode of nutrient solution and change nutrient solution after inoculating 24h, change every other day liquid the last week, change liquid one week rear every day.

8. associational cells model according to claim 6, is characterized in that: in steps d, cultivate 3 days.

9. a method of preparing associational cells model described in claim 1 ~ 8 any one, is characterized in that: it comprises the steps:

A, get Caco-2 cell, recovery, adds in DMEM-20% nutrient solution and cultivates, and by 1:3, goes down to posterity, and cultivates after 2 generations, changes the cultivation that continues to go down to posterity of DMEM-10% nutrient solution into;

B, get and be covered with in the culture dish of mouse tail collagen and 3rd ~ 15 generations prepared by step a generation cell arbitrarily, adjusting cell density is 1.5 * 10 ⁵individual/mL, is inoculated into the culture dish that is covered with mouse tail collagen from AP side, in culture dish BL side, adds DMEM-10% nutrient solution, is placed in 37 ℃, containing 5%CO ₂incubator in cultivate, cultured continuously 17 ~ 25 days, during change nutrient solution, must be loaded with the culture dish of Caco-2 cell model;

C, get Rat Primary Hepatocytes, mix after adding RPMI RPMI-1640, obtain Rat Primary Hepatocytes suspension;

D, get Rat Primary Hepatocytes suspension prepared by step c, adjusting cell density is 1.5 * 10 ⁵individual/ml, is inoculated on the Tissue Culture Plate that is covered with mouse tail collagen, and every hole adds changes 2ml every day liquid, cultivates 1 ~ 5 day, must be loaded with the Tissue Culture Plate of Rat Primary Hepatocytes model;

E, step b gained is loaded with to the culture dish of Caco-2 cell model, is placed on the Tissue Culture Plate that steps d gained is loaded with Rat Primary Hepatocytes model.

10. preparation method according to claim 9, is characterized in that: in described step b:

The culture dish that is covered with mouse tail collagen is prepared as follows: get Millicell suspension type culture dish, adding concentration is the I type mouse tail collagen of 50 μ g/ml, and application of sample amount is 100 μ l/cm ², uv irradiating 1 hour, puts into 37 ℃, 5%CO ₂in incubator, spend the night, with PBS, clean 3 times;

Get the 4th generation cell; Cultured continuously 21 days; Change the mode of nutrient solution and change nutrient solution after inoculating 24h, change every other day liquid the last week, change liquid one week rear every day.

11. preparation methods according to claim 9, is characterized in that: in described steps d, cultivate 3 days.

Described in 12. rights to use requirement 1 ~ 8 any one, the method for associational cells model discrimination medicine, is characterized in that: it comprises the steps:

1. adopt the associational cells model described in claim 1 ~ 8 any one, get medicine to be checked, from the one side dispenser of Caco-2 cell model;

2. collect Rat Primary Hepatocytes model one side sample, detect, according to evaluation medicine to be checked.

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