A cytological detection method for recombinant humanized type III collagen

Abstract

The present invention belongs to the field of biological protein technology, and specifically relates to a cytological detection method for recombinant type III humanized collagen, comprising the following steps: S1 cell culture: inoculating human dermal fibroblasts into a culture medium, and culturing to the logarithmic growth phase for standby use; S2 co-culture: co-culturing different concentrations of standard recombinant type III humanized collagen with human dermal fibroblasts in the logarithmic production phase; S3 constructing a standard curve; S4 quantification and evaluation. The present invention can calculate the concentration of the target protein according to the standard curve, the method is simple and does not require the use of expensive detection instruments, and is more practical and convenient. At the same time, the present invention can batch detect dozens of samples at the same time, greatly improving the detection efficiency. In addition, when the target protein is quantitatively detected in concentration, the biological activity of the target protein can also be evaluated by the cell adhesion rate, which can provide more biological information of the target protein compared to the existing detection methods.

Description

Technical Field

[0001] The invention belongs to the technical field of biological proteins, and in particular relates to a cytological detection method for recombinant type III humanized collagen. Background Art

[0002] In the field of biological protein technology, especially in the fields of cell biology and materials science, the production and application of recombinant proteins is an important research direction. Recombinant proteins are a type of protein synthesized in vivo or in vitro through genetic engineering technology, and are often used to study and treat various diseases. Among them, recombinant humanized type III collagen is an important biomaterial that is widely used in the fields of medicine and bioengineering due to its good biocompatibility and biodegradability.

[0003] There are three main methods for detecting recombinant type III humanized collagen: immunological detection method, large-scale instrument detection method and chemical method. The immunological detection method is to prepare specific antibodies and use immunological methods such as enzyme-linked immunosorbent assay (ELISA) to determine the content of recombinant protein. The large-scale instrument detection method uses large-scale analytical instruments such as high-performance liquid chromatography (HPLC) and mass spectrometer for precise analysis. Chemical methods include Kjeldahl nitrogen determination method, Folin phenol method, etc.

[0004] Although existing technical methods can detect recombinant humanized type III collagen to a certain extent, they all have certain limitations. Immunological detection methods require the preparation of antibodies in advance and the development of kits for determination, which is costly and cumbersome. Large-scale instrument detection methods require the use of high-performance liquid chromatography and mass spectrometers, with high detection thresholds and long method development cycles. Chemical methods, such as the Kjeldahl method and the Folin phenol method, do not have the ability to identify, but only determine the total protein content, and cannot specifically detect the content of recombinant humanized type III collagen. At the same time, none of these existing detection methods can simultaneously evaluate the biological activity of recombinant humanized type III collagen. Therefore, these methods are subject to certain limitations in practical applications, and there is an urgent need to develop a new, simpler, more accurate, and biologically functional detection method. Summary of the invention

[0005] In view of the problems existing in the current detection method of recombinant type III humanized collagen mentioned in the background technology, the present invention provides a cytological detection method of recombinant type III humanized collagen, which can promote the proliferation of human dermal fibroblasts by using recombinant type III humanized collagen, and the proliferation ratio can reflect the biological activity of the collagen peptide segment. The content of the target protein can be calculated according to the standard correspondence between the cell proliferation rate and the concentration of recombinant type III humanized collagen, and the biological activity of the target protein can be evaluated by using the cell adhesion rate of the sample and the standard protein at the same concentration. The specific technical scheme is as follows:

[0006] First, the present invention provides a cytological detection method for recombinant type III humanized collagen, comprising the following steps:

[0007] S1 culture cells: inoculate human dermal fibroblasts into the culture medium and culture them to the logarithmic growth phase for later use;

[0008] S2 co-culture: co-culture different concentrations of standard recombinant humanized type III collagen with human dermal fibroblasts in the logarithmic production phase to obtain co-cultured cells;

[0009] S3 constructs a standard curve: stain the co-cultured cells in S2 with MTT solution and measure their absorbance value, and construct a standard curve A between the absorbance value and the concentration of recombinant humanized type III collagen; stain the co-cultured cells in S2 with fluorescent dye and measure their cell adhesion rate, and construct a standard curve B between the cell adhesion rate and the concentration of recombinant humanized type III collagen;

[0010] S4 quantification and evaluation: The target protein to be detected is first co-cultured with human dermal fibroblasts according to step S2, and then the absorbance value and actual cell adhesion rate Ap are detected using the S3 method. The concentration of the target protein is then calculated using the standard curve A. At the same time, the standard adhesion rate As is calculated using the standard curve B, and the biological activity of the target protein is determined based on Ap / As.

[0011] Invention concept:

[0012] In view of the shortcomings of the existing recombinant humanized type III collagen detection methods, the inventors of the present application creatively combined human dermal fibroblasts that can promote proliferation under a certain concentration of collagen peptides with the detection of recombinant humanized type III collagen. First, recombinant humanized type III collagen was co-cultured with human dermal fibroblasts to promote the proliferation of human dermal fibroblasts. Then, the concentration of human dermal fibroblasts was detected by staining and expressed as absorbance values. By measuring the absorbance values ​​of human dermal fibroblasts co-cultured with recombinant humanized type III collagen at different concentrations, a standard curve of the absorbance values ​​of recombinant humanized type III collagen and human dermal fibroblasts was constructed. In subsequent detections, it is only necessary to detect the absorbance values ​​of human dermal fibroblasts after co-culture with recombinant humanized type III collagen, and the concentration of the target recombinant humanized type III collagen can be calculated using the standard curve.

[0013] At the same time, the adhesion rate of human dermal fibroblasts co-cultured with different concentrations of recombinant humanized type III collagen was detected, and a standard curve of cell adhesion rate and recombinant humanized type III collagen concentration was constructed. When the biological activity of the target protein needs to be evaluated, the actual adhesion rate of the target protein is first detected, and then the standard adhesion rate of the target protein is calculated based on the standard curve, and its biological activity is evaluated based on the ratio of the actual adhesion rate to the standard adhesion rate.

[0014] Furthermore, the culture medium in step S1 is DMEM / F12 culture medium, the serum content in the culture medium is 0.4%, and the culture period also includes the following concentration components: 100U / mL penicillin, 100μg / mL streptomycin and 2mmol / L glutamine; the cell inoculation density is 5×10^4 cells / mL.

[0015] Furthermore, step S2 includes the following steps: adding 100 to 200 human dermal fibroblasts / μl into a test tube, adding recombinant humanized type III collagen thereto, mixing well and culturing for 48 hours to obtain co-cultured cells.

[0016] Furthermore, the step S3 of staining the co-cultured cells in S2 with MTT solution and then determining the absorbance thereof comprises the following steps: adding 5 mg / ml MTT solution to the co-cultured cells obtained in S2, continuing to culture for 4 hours, removing the culture medium and adding dimethyl sulfoxide thereto, shaking until the crystals are fully dissolved, and then measuring the absorbance.

[0017] Furthermore, the standard curve A in step S3 is: y = (AD) / [1+(x / C)^B]+D, wherein y represents the absorbance value, x represents the protein concentration, A=3.57710, B=-1.15211, C=2.17948, D=0.10245.

[0018] Furthermore, the detection range of x in the standard curve A in step S3 is 0.01 mg / mL to 2.56 mg / mL.

[0019] Furthermore, in step S3, the filling machine needs to be sterilized before use, and the sterilization temperature is 121° C. to 135° C. and the time is 20 to 30 minutes.

[0020] Furthermore, in step S3, the co-cultured cells in S2 are stained with a fluorescent dye and then the cell adhesion rate is measured, which includes the following steps: removing the culture medium in the co-cultured cells obtained in S2, adding a fluorescent dye thereto, mixing evenly, sealing, and culturing in the culture phase for 1 hour, observing and recording the initial cell number Nc using a fluorescence microscope, observing and recording the number of adsorbed cells Nt after inverting and centrifuging to fully disperse, and calculating the actual adhesion rate according to Ap=Nt / Nc×100%.

[0021] Furthermore, the standard curve B of adhesion rate and collagen concentration in step S4 is: y= (A - D) / [1 +(x / C)^B] + D, wherein x represents the collagen concentration, y represents the standard adhesion rate, A=72.05052, B=-1.35061, C=0.33493, D=5.82143.

[0022] Furthermore, the criteria for determining the biological activity of the recombinant humanized type III collagen in step S4 are:

[0023] If Ap / As ≥ 2, the biological activity is judged to be strong;

[0024] If 1≤Ap / As<2, the biological activity is judged to be strong;

[0025] If 0.5<Ap / As<1, the biological activity is judged to be weak;

[0026] If Ap / As < 0.5, the biological activity is judged to be weak.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] 1) The present invention first constructs a standard curve of recombinant type III humanized collagen and human dermal fibroblast concentration (expressed by absorbance value) through standard protein. During detection, it is only necessary to co-culture the target protein with human dermal fibroblasts and then detect the concentration of human dermal fibroblasts to calculate the concentration of the target protein according to the standard curve. The method is simple and does not require the use of expensive detection instruments. Compared with immunological detection methods and large-scale instrument detection methods, it has a low threshold for use and is more practical and convenient. At the same time, the human dermal fibroblasts used in the present invention are easy to obtain and have low culture costs. When detecting, taking a 96-well plate as an example, dozens of samples can be batch-tested at the same time, which greatly improves detection efficiency.

[0029] 2) The present invention uses a protein expressed in the core functional region of human type III collagen when constructing a standard curve. The protein has biological activity for cell proliferation and is specific for the detection of recombinant humanized type III collagen. Compared with chemical methods such as the Kjeldahl method and the Folin-phenol method, the protein has higher identification ability and accuracy.

[0030] 3) The present invention also constructs a standard curve of human dermal fibroblast cell adhesion rate and recombinant type III humanized collagen concentration. When the concentration of the target protein is quantitatively detected, the biological activity of the target protein can also be evaluated by the cell adhesion rate. Compared with the existing detection methods, it can provide more biological information of the target protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is the fitting diagram of the standard curve A in the present invention;

[0032] Figure 2 It is the fitting diagram of the standard curve B in the present invention. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solution and advantages of the present invention more clear, the technical solution of the present invention will be clearly and completely described below in conjunction with embodiments. Example

[0034] A cytological detection method for recombinant type III humanized collagen comprises the following steps:

[0035] S1 culture cells: inoculate human dermal fibroblasts into the culture medium and culture them to the logarithmic growth phase for later use;

[0036] Specifically, prepare DMEM / F12 culture medium, in which the serum content is 0.4%, and other components include penicillin (100U / mL), streptomycin (100μg / mL) and glutamine (2mmol / L). Inoculate human dermal fibroblasts (HSF) into the culture medium at a density of 5×10^4 cells / mL and culture in an incubator at 37°C and 5% CO2 until the logarithmic growth phase.

[0037] S2 co-culture: co-culture different concentrations of standard recombinant humanized type III collagen with human dermal fibroblasts in the logarithmic production phase to obtain co-cultured cells;

[0038] Specifically, a commercially available recombinant protein expressed in the core functional region of human type III collagen was used as a standard substance and diluted to an appropriate concentration with sterile PBS (pH = 7.4) buffer. HSF cells in the logarithmic phase were collected and 50 μL was added to each well of a 96-well plate, with 5000-10000 cells per well. The standard protein solution was added to the culture medium to a final concentration of 0.0 mg / mL, 0.01 mg / mL, 0.04 mg / mL, 0.16 mg / mL, 0.64 mg / mL and 2.56 mg / mL, respectively. Standard protein culture solutions of different concentrations were respectively drawn and added to a 96-well plate. At the same time, at least 2 dilution groups of samples to be tested were set up, with 3 replicates for each concentration, and cultured in a cell culture incubator for 48 hours.

[0039] S3 constructs a standard curve: stain the co-cultured cells in S2 with MTT solution and measure their absorbance value, and construct a standard curve A between the absorbance value and the concentration of recombinant humanized type III collagen; stain the co-cultured cells in S2 with fluorescent dye and measure their cell adhesion rate, and construct a standard curve B between the cell adhesion rate and the concentration of recombinant humanized type III collagen;

[0040] Specifically, add 5 mg / mL MTT solution to each well of the 96-well plate of S2 and continue to culture for 4 hours. After the culture is completed, carefully remove the culture medium in the well, add 150 μL of dimethyl sulfoxide to each well, place it on a shaker and shake at a low speed for 10 minutes to fully dissolve the crystals, measure the absorbance of each well at OD490nm on the microplate reader, and set the zero well STD0 (containing culture medium, MTT, dimethyl sulfoxide) at the same time. The absorbance value after deducting the blank well (0.0 mg / mL) is shown in Table 1 below:

[0041] According to the absorbance value of the sample group to be tested, the content of recombinant type III humanized collagen was calculated by substituting the standard curve into the standard curve. With the concentration of the standard protein solution as the horizontal axis and the absorbance value as the vertical axis, the standard curve A was obtained by fitting the standard curve using four parameters: y = (AD) / [1+(x / C)^B]+D, where y represents the absorbance value, x represents the protein concentration, A=0.93976, B=-0.66206, C=3.11610, and D=0.09369. The fitting diagram of the standard curve is shown in the attached figure. Figure 1 As shown (the vertical axis absorbance value represents the absorbance value), the r^2 of the fitted standard curve A is 0.99969.

[0042] When evaluating biological activity, discard the cell culture medium in the 96-well plate, add 200μL of cell culture medium containing 10% fluorescent dye, cover with aluminum foil, and incubate in a 37℃, 5% CO2 incubator for 1h. Cover the well plate with a sealing film, observe and record the initial number of cells (Nc) with an inverted fluorescence microscope, centrifuge at 1000rpm for 5min, discard the sealing film and supernatant, wash once with PBS buffer, add 100μL of washing solution, and count the adsorbed cells (Nt), adhesion rate = Nt / Nc×100%. The test results are shown in Table 2 below:

[0043] With the concentration of the standard protein solution as the horizontal axis and the adhesion rate as the vertical axis, the standard curve B was obtained by four-parameter fitting: y= (A - D) / [1 + (x / C)^B]+ D, where x represents the collagen concentration, y represents the standard adhesion rate, A=72.05052, B=-1.35061, C=0.33493, D=5.82143. The fitting diagram of the standard curve B is shown in the attached figure. Figure 2 As shown (the vertical axis adhesion rate represents the standard adhesion rate), the r^2 of standard curve B is 0.99980.

[0044] S4 quantification and evaluation: The target protein to be detected is first co-cultured with human dermal fibroblasts according to step S2, and then the absorbance value and actual cell adhesion rate Ap are detected using the S3 method. The concentration of the target protein is then calculated using the standard curve A. At the same time, the standard adhesion rate As is calculated using the standard curve B, and the biological activity of the recombinant type III humanized collagen is determined based on Ap / As.

[0045] Specifically, if there is a target protein that needs to be detected, the target protein is first diluted into three or more concentrations and then mixed with human dermal fibroblasts in the logarithmic production period, cultured according to the method of step S2, and then the absorbance value is detected according to the method of step S3, and the concentration of the target protein is calculated according to the standard curve A. When biological evaluation is required, the actual adhesion rate Ap of the target protein is first calculated according to the method of step S3, and then the standard adhesion rate As is calculated by the standard curve B according to the concentration of the target protein, and the biological activity of the target protein is determined according to Ap / As. The judgment standard is that if Ap / As≥2, the biological activity is judged to be strong; if 1≤Ap / As<2, the biological activity is judged to be relatively strong; if 0.5<Ap / As<1, the biological activity is judged to be relatively weak; if Ap / As<0.5, the biological activity is judged to be weak.

[0046] Experimental example

[0047] The content and biological activity of recombinant type III humanized collagen in a certain manufacturer's recombinant collagen freeze-dried powder were determined. The recombinant collagen concentrations were diluted to 0.1 mg / mL, 0.5 mg / mL, and 1.0 mg / mL according to the product label content, and three parallel wells were set for each concentration. The content test results are shown in Table 3 below:

[0048]

[0049] The results of the biological activity test are shown in Table 4 below:

[0050]

[0051] It is obvious to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be regarded as exemplary and non-restrictive from any point of view. In addition, it should be understood that although this specification is described in accordance with the embodiments, it does not contain only one technical solution. This narrative of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in the embodiments can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A cytological detection method for recombinant type III humanized collagen, characterized in that: The following steps are involved: S1 culture cells: inoculate human dermal fibroblasts into the culture medium and culture them to the logarithmic growth phase for later use; S2 co-culture: co-culture different concentrations of standard recombinant humanized type III collagen with human dermal fibroblasts in the logarithmic production phase to obtain co-cultured cells; S3 constructs a standard curve: stain the co-cultured cells in S2 with MTT solution and measure their absorbance value, and construct a standard curve A between the absorbance value and the concentration of recombinant humanized type III collagen; stain the co-cultured cells in S2 with fluorescent dye and measure their cell adhesion rate, and construct a standard curve B between the cell adhesion rate and the concentration of recombinant humanized type III collagen; S4 quantification and evaluation: The target protein to be detected is first co-cultured with human dermal fibroblasts according to step S2, and then the absorbance value and actual cell adhesion rate Ap are detected using the method of S3, and then the concentration of the target protein is calculated using the standard curve A; the standard adhesion rate As is calculated using the standard curve B according to the target protein concentration, and the biological activity of the target protein is determined according to Ap / As; The step S3 of staining the co-cultured cells in S2 with a fluorescent dye and then measuring the cell adhesion rate comprises the following steps: removing the culture medium in the co-cultured cells obtained in S2, adding the fluorescent dye thereto, mixing evenly, sealing, and culturing in the culture phase for 1 hour, observing and recording the initial cell number Nc using a fluorescent microscope, observing and recording the adsorbed cell number Nt after inverting and centrifuging to fully disperse, and calculating the adhesion rate according to adhesion rate = Nt / Nc×100%.

2. The cytological detection method of recombinant humanized type III collagen according to claim 1, characterized in that: The culture medium in step S1 is DMEM / F12 culture medium, the serum content in the culture medium is 0.4%, and the culture medium also includes the following concentration components: 100U / mL penicillin, 100μg / mL streptomycin and 2mmol / L glutamine; the cell inoculation density is 5×10^4 cells / mL.

3. The cytological detection method of recombinant humanized type III collagen according to claim 2, characterized in that: The step S2 includes the following steps: adding 100 to 200 / μl human dermal fibroblasts into a test tube, adding recombinant type III humanized collagen thereto, mixing evenly and culturing for 48 hours to obtain co-cultured cells.

4. The cytological detection method of recombinant humanized type III collagen according to claim 3, characterized in that: The step S3 uses MTT solution to stain the co-cultured cells in S2 and then determines the absorbance value thereof, comprising the following steps: adding 5 mg / ml MTT solution to the co-cultured cells obtained in S2, continuing to culture for 4 hours, removing the culture medium and adding dimethyl sulfoxide thereto, shaking until the crystals are fully dissolved, and then measuring the absorbance value.

5. The cytological detection method of recombinant humanized type III collagen according to claim 4, characterized in that: The standard curve A in step S3 is: y = (AD) / [1+(x / C)^B]+D, where y represents the absorbance value, x represents the protein concentration, A=3.57710, B=-1.15211, C=2.17948, and D=0.10245.

6. The cytological detection method of recombinant humanized type III collagen according to claim 5, characterized in that: The detection range of x in the standard curve A in step S3 is 0.01 mg / mL to 2.56 mg / mL.

7. A cytological detection method for recombinant humanized type III collagen according to claim 6, characterized in that: The standard curve B of adhesion rate and collagen concentration in step S3 is y= (A - D) / [1 + (x / C)^B] +D, where x represents collagen concentration, y represents standard adhesion rate, A=72.05052, B=-1.35061, C=0.33493, and D=5.82143.

8. According to the cytological detection method of recombinant humanized type III collagen as described in claim 7, the criterion for determining the biological activity of the recombinant humanized type III collagen in step S4 is: If Ap / As ≥ 2, the biological activity is judged to be strong; If 1≤Ap / As<2, the biological activity is judged to be strong; If 0.5<Ap / As<1, the biological activity is judged to be weak; If Ap / As < 0.5, the biological activity is judged to be weak.

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