CN103131632A - Screening method for proteinuria component induced renal interstitial fibrosis based on micro-fluidic chip - Google Patents
Screening method for proteinuria component induced renal interstitial fibrosis based on micro-fluidic chip Download PDFInfo
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- CN103131632A CN103131632A CN2011103868351A CN201110386835A CN103131632A CN 103131632 A CN103131632 A CN 103131632A CN 2011103868351 A CN2011103868351 A CN 2011103868351A CN 201110386835 A CN201110386835 A CN 201110386835A CN 103131632 A CN103131632 A CN 103131632A
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Abstract
The invention provides a screening method for proteinuria component induced renal interstitial fibrosis based on a micro-fluidic chip. The micro-fluidic chip comprises twelve identical structure units. The structure units are connected through central pools. Each structure unit is composed of a stimulant inlet pool (1), a fluid buffer channel (2), a cell culture pool (3) and a central pool (4). The screening method for the proteinuria component induced renal interstitial fibrosis based on the micro-fluidic chip integrates chip cell culture, cell and factor interaction, fluid stimulation, cell marking and cell responding signal detection on the chip of several square centimeters, and can be used for analyzing a proteinuria component induced renal interstitial fibrosis mechanism. Compared with a perforated plate technology, the screening method for the proteinuria component induced renal interstitial fibrosis based on the micro-fluidic chip saves the cumbersome step of repeated cell inoculation, solves the problems that different factors cannot act on cells at the same time under the flow condition, reduces the consumption of the cells and reagent, and can achieve a plurality of experiment parameters through one-time operation.
Description
Technical field
The invention belongs to the micro-fluidic chip technology is applied to the biomedical research field, be specifically related to a kind of screening method that causes kidney region fibrosis based on the multiple protein urine component of micro-fluidic chip.
Background technology
Uremia is harm people's life and healthy major disease, and its treatment cost is expensive, all brings heavy economical load for individual, society.At present in China, various primary glomerulopathies remain and cause uremic first reason, and the proteinuria that continues not alleviate is that above-mentioned progression of disease is to uremic independent hazard factor, greatly reason be because proteinuria can damage renal cells (Proximal tubular epithelial cell, PTEC) and between matter and then cause kidney region fibrosis.Therefore, resolve proteinuria wherein various ingredients the kidney region fibrosis mechanism that causes, seek to prevent means significant to reducing the uremia crowd.
Although proteinuria causes kidney region fibrosis and has obtained widespread consensus, but most research is all for single factors such as albumin, IgG, damage, apoptosis, a matter inflammatory reaction of renal tubular epithelial etc. inquired in the stimulations such as complement C3, and the operating method that major part is based on this static state of array microwell plate is carried out cell cultures, applies stimulation in orifice plate.In true internal milieu, the liquid in uriniferous tubules is to be in flow state very slowly, so the albumen in crude urine is to be in the utmost point to stimulate PTEC under flow state slowly.So, we more expect to set up one more near the renal cells in-vitro culture model of internal milieu, add simultaneously multiple stimulus on this model, investigate above-mentioned factor and under flow state, critical event PTEC inflammation, apoptosis and epithelium in a kidney region fibrosis-matter is transformed (epithelial-mesenchymal transition, EMT) impact of EMT can disclose the mechanism that proteinuria causes kidney region fibrosis better.
The micro-fluidic chip technology that grew up in recent years provides possibility for the realization of our imagination.Microfluid based Lab on a chip claims again chip lab (Lab-on-a-Chip) or micro-fluidic chip (Microfluidic), refer to related sample preparation, the reaction in fields such as chemistry and biology, separate, the basic operation units such as detection, cell cultures, sorting, cracking are integrated or substantially be integrated on the chip of more than square centimeters (even less), form network by the microchannel, run through whole system with controlled fluid, in order to a kind of technology of the various functions that replace conventional chemical or biology laboratory.The essential characteristic of Microfluid based Lab on a chip and sharpest edges are that multiple monotechnics flexible combination, scale on the controlled small platform of integral body are integrated.Develop so far, the micro-fluidic chip technology has begun to be applied in the different field of life science, and has become one of important technological platform in systems biology research.Particularly in recent years, due to multidimensional network feature and the function integration characteristic of micro-fluidic chip, might make that conventional cell cultures, cell are excited, the process integration such as cell marking and detection complete on a very little chip.For PTEC, can realize multifactorly under identical conditions simultaneously cell being stimulated by micro-fluidic chip in theory, make it the pathology environment of more approaching reality.
Summary of the invention
The purpose of this invention is to provide a kind of screening method that causes kidney region fibrosis based on the proteinuria component of micro-fluidic chip, how the method can realize the characteristics that multifactor stimulation is carried out simultaneously under flow state if having, also easily realize the behavior of Real-Time Monitoring cellular response.
The invention provides a kind of micro-fluidic chip, this chip is made of 12 identical structural units, these identical structural units are connected by the pond, center, and each structural unit is by stimulator entrance pond (1), fluid cushion passage (2), cell culture insert (3) and pond, center (4) formation.
Micro-fluidic chip provided by the invention, described micro-fluidic chip is formed by the two-layer irreversible sealing-in in up and down, and upper layer of material is the PDMS polymkeric substance, and subsurface material is glass.
Micro-fluidic chip provided by the invention after the irreversible sealing-in of described micro-fluidic chip, adds 50 μ g/ml NTxes to carry out finishing to chip, to be conducive to cell attachment from the pond, center.
Micro-fluidic chip provided by the invention, fluid cushion passage (2) in described chip is the design of winding type, on the one hand when guaranteeing the cell application of sample, cell can rest on cell culture chamber preferably, and incoming fluid buffer channel (2) not substantially, to be in order guaranteeing to add induction factor in stimulator entrance pond (1) on the other hand, the situation of series fluid flow not to occur between a plurality of structural units.
The present invention also provides a kind of screening method that causes kidney region fibrosis based on the proteinuria component of described micro-fluidic chip, and procedure is as follows:
After cells in vitro digestion, add fresh medium, with pipettor, cell suspension is added and be positioned at the chip center pond, chip is placed in microscopically and observes, when seeing that under mirror cell after flowing into cell culture chamber under the effect of static pressure fluid, is covered in chip surface with the 5-10ml fresh medium rapidly, in chip, cell is no longer mobile immediately, due to the settlement action of cell self gravitation, cell can be parked in cell culture chamber very soon;
Chip is put into 37 ℃ of incubator static cultivation, approximately take out chip after 30min-2h, siphon away the unnecessary nutrient solution of chip surface, and then chip is put back to continued in 37 ℃ of incubators to cultivate;
After cell attachment, pond, center and syringe pump are coupled together by four fluorine tube, open syringe pump, regulating flow velocity is 0.6 μ l/min, sequentially add the different induction factor of equal volume from stimulator entrance pond, due to the effect of taking out of syringe pump, the liquid that makes chip moderate stimulation thing entrance pond is the incoming fluid buffer channel gradually, then flows to cell culture chamber; After 12 ~ 72h, chip is placed in microscopically carries out the light field photograph taking, the change of observation of cell form;
Disconnect being connected of pond, center and syringe pump after 72h, then add fixating reagent, washing soln, staining agent etc. in the pond, center, under static pressure drove, fluid inflow cell culture chamber carried out cell dyeing; At last, micro-fluidic chip is placed under fluorescent microscope, carries out cell to the detection of induction factor response parameter.
The present invention has following advantage:
1. hormesis when the present invention can realize multiple protein urine component to renal cells, and there is certain surge time between different induction factors when flowing through the fluid cushion passage, can not cause mutual interference effect occurs between different induction factors, can obtain simultaneously the information of 12 groups of cellular response parameters.
2. the present invention can add respectively cell suspension, fixed solution, washing soln and label solution just can complete inoculation, washing and the marking operation of cell in cell culture chamber from the pond, center.
3. compare with traditional porous plate technology, the present invention has reduced the consumption of cell and reagent significantly, has built one and more has been used for the research kidney region fibrosis near the model of pathology environment in body.
Description of drawings
In Fig. 1, (a) is micro-fluidic chip one-piece construction schematic diagram, (b) is the enlarged view of one of them structural unit;
Fig. 2 is after transforming growth factor TGF-β stimulates the HK-2 cell, at different time (0h, 24h, 48h, 72h), and the cell light field photo under different concns (0,2.5ng/ml, 5ng/ml, 10ng/ml) condition;
Fig. 3 is after transforming growth factor TGF-β stimulates HK-2 cell 72h, different concns (0,2.5ng/ml, 5ng/ml, 10ng/ml) iuntercellular under the condition connects (E-cadherin), fibroblast-like cell specific albumen (FSP-1), smooth muscle actin (the immunofluorescence picture that α-SMA) detects;
Fig. 4 is after transforming growth factor TGF-β stimulates HK-2 cell 72h, the E-cadherin under different concns (0,2.5ng/ml, 5ng/ml, 10ng/ml) condition, FSP-1, the data analysis figure of the fluorescence intensity that α-SMA detects;
Fig. 5 is after the Healthy Human Serum (inactive human health serum, ihhs) of deactivation stimulates the HK-2 cell, at different time (0h, 24h, 48h, 72h), and the cell light field photo under different concns (0,1%, 3%, 5%) condition;
Fig. 6 is after the Healthy Human Serum (ihhs) of deactivation stimulates HK-2 cell 72h, the E-cadherin under different concns (0,1%, 3%, 5%) condition, FSP-1, the immunofluorescence picture that α-SMA detects;
Fig. 7 is after the Healthy Human Serum (ihhs) of deactivation stimulates HK-2 cell 72h, the E-cadherin under different concns (0,1%, 3%, 5%) condition, FSP-1, the data analysis figure of the fluorescence intensity that α-SMA detects;
Fig. 8 is after the Healthy Human Serum (human health serum, hhs) of not deactivation stimulates the HK-2 cell, at different time (0h, 24h, 48h, 72h), and the cell light field photo under different concns (0,1%, 3%, 5%) condition;
Fig. 9 is after the Healthy Human Serum (hhs) of not deactivation stimulates HK-2 cell 72h, the E-cadherin under different concns (0,1%, 3%, 5%) condition, FSP-1, the immunofluorescence picture that α-SMA detects;
Figure 10 is after the Healthy Human Serum (hhs) of not deactivation stimulates HK-2 cell 72h, the E-cadherin under different concns (0,1%, 3%, 5%) condition, FSP-1, the data analysis figure of the fluorescence intensity that α-SMA detects;
Figure 11 is after the Healthy Human Serum of deactivation and the complement C 3 of 10nM (ihhs+C3a) stimulate the HK-2 cell, at different time (0h, 24h, 48h, 72h), and the cell light field photo under different concns (0,1%, 3%, 5%) condition;
Figure 12 is after the Healthy Human Serum of deactivation and the complement C 3 of 10nM (ihhs+C3a) stimulate HK-2 cell 72h, the E-cadherin under different concns (0,1%, 3%, 5%) condition, FSP-1, the immunofluorescence picture that α-SMA detects;
Figure 13 is after the Healthy Human Serum of deactivation and the complement C 3 of 10nM (ihhs+C3a) stimulate HK-2 cell 72h, the E-cadherin under different concns (0,1%, 3%, 5%) condition, FSP-1, the data analysis figure of the fluorescence intensity that α-SMA detects;
Figure 14 is after complement C 3 stimulates the HK-2 cell, at different time (0h, 24h, 48h, 72h), and the cell light field photo under different concns (0,1nM, 5nM, 10nM) condition;
Figure 15 is after complement C 3 stimulates HK-2 cell 72h, the E-cadherin under different concns (0,1nM, 5nM, 10nM) condition, FSP-1, the immunofluorescence picture that α-SMA detects;
Figure 16 is after complement C 3 stimulates HK-2 cell 72h, the E-cadherin under different concns (0,1nM, 5nM, 10nM) condition, FSP-1, the data analysis figure of the fluorescence intensity that α-SMA detects.
Embodiment
Following examples will be further described the present invention, but not thereby limiting the invention.
At first carry out the inoculation of HK-2 cell:
After uriniferous tubules near-end epithelial cell (human proximal tubular epithelial cell, HK-2) digestion, add fresh medium, adjusting cell density is 1 * 10
6/ ml, with pipettor, cell suspension is added the pond, center of chip as shown in Figure 1, when observing cell after flowing into cell culture chamber under the effect of static pressure fluid, rapidly the 5-10ml fresh medium is covered in chip surface, in chip, the HK-2 cell is no longer mobile immediately, settlement action due to the cell self gravitation, cell can be parked in cell culture chamber very soon, chip is put into 37 ℃ of incubator static cultivation, approximately take out chip after 30min-2h, siphon away the unnecessary nutrient solution of chip surface, and then chip is put back to continued in 37 ℃ of incubators to cultivate.
Then carry out the application of sample of induction factor: pond, center and syringe pump are coupled together by four fluorine tube, open syringe pump, regulating flow velocity is 0.6 μ l/min, and sequentially add the induction factor of equal volume from stimulator entrance pond: 1-3 does not add any stimulus, as negative control group; 4-6 adds respectively 2.5ng/ml, 5ng/ml, and the transforming growth factor of 10ng/ml (transforming growth factor-β, TGF-β) is as positive controls; 7-9 adds respectively 1%, 3%, 5% Healthy Human Serum (human health serum, hhs); 10-12 adds respectively the Healthy Human Serum (inactive human health serum, ihhs) of 1%, 3%, 5% deactivation, after application of sample is completed, chip is put back in incubator.At 0h, 24h, 48h, 72h are placed in microscopically and carry out the light field photograph taking, the change of observation of cell form respectively.
Carry out at last cell dyeing: disconnect being connected of pond, center and syringe pump after 72h, then add fixating reagent 4% paraformaldehyde, phosphate buffered saline(PBS) (phosphate buffer saline, PBS), goat sealing serum, corresponding primary antibodie reagent (the anti-human E-calcium of rabbit conglutnin E-cadherin in the pond, center; The anti-human desmocyte sample of rabbit specific proteins FSP-1; Mouse-anti human smooth muscle Actin muscle α-SMA), the two anti-reagent (goat anti-rabbit iggs of rhodamine B mark; The goat anti-mouse IgG of FITC mark etc., under static pressure drove, fluid flowed into cell culture chamber cell is carried out cell dyeing; At last, micro-fluidic chip is placed under fluorescent microscope, carries out cell to the detection of induction factor response parameter.
Result shows: compare with negative control group, transforming growth factor (TGF-β) can significantly induce HK-2 cell generation form to change under the concentration conditions of 5ng/ml, distribution by the paving stone sample becomes spindle shape (Fig. 2), become simultaneously fiber-like specific proteins FSP-1, significantly raising appears in the expression amount of smooth muscle actin α-SMA; The significantly downward of expression amount appearance of calcium conglutnin E-caherin (Fig. 3-Fig. 4); These results comprehensively illustrate, TGF-β has multifunctional bio and learns active cytokine as a kind of, really plays a crucial role in bringing out epithelium-matter conversion process (epithelial-mesenchymal transition, EMT).Find simultaneously, Healthy Human Serum hhs has also occurred similarly inducing effect (Fig. 8-Figure 10) with TGF-β when stimulating the HK-2 cell.And the Healthy Human Serum of deactivation does not cause the change of cellular form when stimulation HK-2 cell, and the also not obviously change of generation of corresponding EMT mark (Fig. 5-Fig. 7).This prompting: the complement component of losing in the inactivation process of Healthy Human Serum can exert an influence to the EMT process of inducing the HK-2 cell.
The inoculation of HK-2 cell and staining procedure are with " embodiment 1 "
Concrete induction factor application of sample process is: pond, center and syringe pump are coupled together by four fluorine tube, open syringe pump, regulating flow velocity is 0.6 μ l/min, and sequentially add the induction factor of equal volume from stimulator entrance pond: 1-3 does not add any stimulus, as negative control group; 4-6 adds respectively 2.5ng/ml, 5ng/ml, and the transforming growth factor of 10ng/ml (transforming growth factor-β, TGF-β) is as positive controls; 7-9 adds respectively the ihhs that contains different concns (1%, 3%, 5%) and the mixture of complement C 3 (10nM); 10-12 adds respectively the Healthy Human Serum (inactive human health serum, ihhs) of 1%, 3%, 5% deactivation, after application of sample is completed, chip is put back in incubator.At 0h, 24h, 48h, 72h are placed in microscopically and carry out the light field photograph taking, the change of observation of cell form respectively.
Result shows: complement-fixing C3a concentration is 10nM, the Healthy Human Serum ihhs concentration of adjusting deactivation changes (1%, 3%, 5%), under these conditions, obvious form all can occur and change in the HK-2 cell, and (become fiber-like specific proteins FSP-1, significantly raising appears in the expression amount of smooth muscle actin α-SMA to be accompanied by the corresponding change of EMT mark generation; Significantly lowering appears in the expression amount of calcium conglutnin E-caherin), see Figure 11-Figure 13.And dependency (Figure 14-Figure 16) of concentration and time has also appearred in the complement C 3 of different concns (1nM, 5nM, 10nM) when stimulating the HK-2 cell.This explanation confirms that through the investigation of above-mentioned experiment the complement component of losing to renal cells, the EMT process occurs and plays a key effect in to the Healthy Human Serum inactivation process, played the part of important role in the kidney region fibrosis process.
Claims (6)
1. micro-fluidic chip, it is characterized in that: this chip is made of 12 identical structural units, these identical structural units are connected by the pond, center, and each structural unit is by stimulator entrance pond (1), fluid cushion passage (2), cell culture insert (3) and pond, center (4) formation.
2. according to micro-fluidic chip claimed in claim 1, it is characterized in that: described micro-fluidic chip is formed by the two-layer irreversible sealing-in in up and down, and upper layer of material is the PDMS polymkeric substance, and subsurface material is glass.
3. according to micro-fluidic chip claimed in claim 1, it is characterized in that: after the irreversible sealing-in of described micro-fluidic chip, add NTx from the pond, center.
4. according to micro-fluidic chip claimed in claim 3, it is characterized in that: described NTx concentration is 50 μ g/ml.
5. according to micro-fluidic chip claimed in claim 1, it is characterized in that: the fluid cushion passage (2) in described chip is the structure design of winding type.
6. cause the screening method of kidney region fibrosis based on the proteinuria component of the described micro-fluidic chip of claim 1, it is characterized in that: procedure is as follows:
After cells in vitro digestion, add fresh medium, with pipettor, cell suspension is added the chip center pond, chip is placed in microscopically and observes, when seeing that under mirror cell after flowing into cell culture chamber under the effect of static pressure fluid, is covered in chip surface with the 5-10ml fresh medium rapidly, in chip, cell is no longer mobile immediately, due to the settlement action of cell self gravitation, cell can be parked in cell culture chamber very soon;
Chip is put into 37 ℃ of incubator static cultivation, approximately take out chip after 30min-2h, siphon away the unnecessary nutrient solution of chip surface, and then chip is put back to continued in 37 ℃ of incubators to cultivate;
After cell attachment, pond, center and syringe pump are coupled together by four fluorine tube, open syringe pump, regulating flow velocity is 0.6 μ l/min, sequentially add the different induction factor of equal volume from stimulator entrance pond, due to the effect of taking out of syringe pump, the liquid that makes chip moderate stimulation thing entrance pond is the incoming fluid buffer channel gradually, then flows to cell culture chamber; After 12 ~ 72h, chip is placed in microscopically carries out the light field photograph taking, the change of observation of cell form;
Disconnect being connected of pond, center and syringe pump after 72h, then add fixating reagent, washing soln, staining agent etc. in the pond, center, under static pressure drove, fluid inflow cell culture chamber carried out cell dyeing; At last, micro-fluidic chip is placed under fluorescent microscope, carries out cell to the detection of induction factor response parameter.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104178423A (en) * | 2014-08-26 | 2014-12-03 | 东南大学 | Micro-fluidic- technique-based living cell culture device |
| CN107955781A (en) * | 2016-10-14 | 2018-04-24 | 中国科学院大连化学物理研究所 | The liver of metabolic process-kidney system in aids drug body based on micro-fluidic chip |
| CN108148752A (en) * | 2016-12-06 | 2018-06-12 | 中国科学院大连化学物理研究所 | A kind of integrated drug screening and colouring method based on micro-fluidic chip |
-
2011
- 2011-11-29 CN CN2011103868351A patent/CN103131632A/en active Pending
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| 刘伏友: "蛋白尿致肾小管间质纤维化的机制及防治", 《中华肾脏病杂志》 * |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104178423A (en) * | 2014-08-26 | 2014-12-03 | 东南大学 | Micro-fluidic- technique-based living cell culture device |
| CN104178423B (en) * | 2014-08-26 | 2016-04-13 | 东南大学 | A kind of active somatic cell culture apparatus based on microflow control technique |
| CN107955781A (en) * | 2016-10-14 | 2018-04-24 | 中国科学院大连化学物理研究所 | The liver of metabolic process-kidney system in aids drug body based on micro-fluidic chip |
| CN107955781B (en) * | 2016-10-14 | 2021-03-09 | 中国科学院大连化学物理研究所 | Liver-kidney system for simulating in-vivo metabolic process of medicine based on micro-fluidic chip |
| CN108148752A (en) * | 2016-12-06 | 2018-06-12 | 中国科学院大连化学物理研究所 | A kind of integrated drug screening and colouring method based on micro-fluidic chip |
| CN108148752B (en) * | 2016-12-06 | 2023-09-26 | 中国科学院大连化学物理研究所 | Integrated drug screening and dyeing method based on microfluidic chip |
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Application publication date: 20130605 |