WO2018192156A1

WO2018192156A1 - Ultra-remote intelligent diagnosis and treatment system for diabetes

Info

Publication number: WO2018192156A1
Application number: PCT/CN2017/100448
Authority: WO
Inventors: 叶海峰; 邵佳伟; 余贵玲; 薛帅; 于袁欢; 白郁
Original assignee: 苏州欣赛生物科技有限公司
Priority date: 2017-04-21
Filing date: 2017-09-05
Publication date: 2018-10-25

Abstract

An ultra-remote intelligent diagnosis and treatment system for diabetes comprises an in-vivo implantable LED and a hydrogel transplant carrier containing a photo-controlled engineered cell, which is formed from a hydrogel-coated engineered cell regulated and expressed by far-red light and secreting insulin or glucagon-like peptide, and a far-infrared LED. A far-red light-regulated gene expression loop control system comprising a photoreceptor to receive a far-red light source, a processor to process a signal transmitted by the photoreceptor, and an effector to respond to the signal transmitted by the processor is further provided. The far-red light-regulated gene expression loop control system controls the level of gene expression through the far-red light-regulated gene expression. The system can convert a blood glucose concentration signal into an optical signal or control command of a user's mobile phone to control an optical module implanted in-vivo to precisely regulate the expression level of insulin or glucagon-like peptide, thereby achieving a goal of an automatic, precise, and individualized treatment of diabetes.

Description

Diabetes ultra-long-range intelligent diagnosis and treatment system

Technical field

The invention relates to a multidisciplinary field of electronic engineering, synthetic biology and optogenetics, and particularly relates to a diabetes ultra-long-range intelligent diagnosis and treatment system for diagnosing and treating diabetes by integrating an optical module in vivo, and a construction method thereof Application in the treatment of diabetes.

Background technique

In the field of synthetic biology and disease treatment, molecular switches that artificially regulate gene expression have become an indispensable means in the treatment of diseases. At present, there are many systems in the world that induce gene expression by artificial regulation, and induce gene expression systems by chemical inducers or physical methods.

Light is an ideal inducer of gene expression. It is ubiquitous in nature, readily available, has space-time specificity, and is non-toxic. Therefore, the use of light as an inducer to regulate gene expression and to treat various diseases is of great application value.

At present, the treatment of diabetes mainly includes insulin injection, taking drugs and controlling diet. However, the current medical level still cannot completely cure diabetes. Diabetes patients need daily oral hypoglycemic drugs or insulin to maintain blood sugar stability, and insulin injection cannot be achieved. Controlled release of insulin can easily cause a risk of hypoglycemia. Therefore, it is very important to be able to accurately monitor the blood glucose concentration in the body and to accurately adjust the blood glucose concentration in time for the treatment of diabetes. At present, there are a few intelligent blood glucose monitors on the market, which can only give the blood sugar value simply, and the degree of intelligence is not high. The patient still needs to adjust the treatment plan and living habit according to the given blood sugar value, and has not been able to collect the diagnosis and The treatment of diabetes is an ultra-long-range intelligent diagnosis and treatment system. There is an urgent need to seek new treatment models to improve the treatment, reduce the risk of treatment, and improve the convenience of treatment.

Summary of the invention

The technical problem to be solved by the present invention is to address the deficiencies of the above prior art, and to provide a diabetes ultra-long-range intelligent diagnosis and treatment system which integrates diagnosis and treatment of diabetes by integrating an optical module in vivo, and the intelligent diagnosis and treatment system is in the treatment of diabetes. The application of the invention is in the optical module of the invention, and the blood glucose concentration feedback regulation in the receptor and the direct remote control of the user's mobile phone are directly controlled, and the invention has the advantages of ultra-remote control, accurate diagnosis of diabetes, rapid treatment, simple operation and high intelligence. The treatment of good insulation and non-toxic side effects can achieve the purpose of automation, precision and individualized treatment of diabetes. It has great potential application value in the diagnosis and treatment of artificial intelligence customized cells and diseases, and can be widely used in clinical in the future. treatment.

The invention provides a diabetes ultra-long-range intelligent diagnosis and treatment system, the device comprises: an automatic blood sugar data control system, a blood glucose data remote control system, a power supply module (such as a wireless power supply module), and an optical module; the blood glucose data automatic control system includes a blood glucose concentration detecting system (or a blood glucose concentration detecting device) and a blood sugar data processing unit; the blood sugar data automatic control system generates blood glucose concentration data by acquiring the blood glucose concentration value by the blood glucose concentration detector; the blood glucose data processing unit Extracting a blood glucose concentration value from the blood glucose concentration data, and outputting a current corresponding to the voltage to the wireless power supply module according to the blood glucose concentration value; the blood glucose data remote control system includes a mobile device and an intelligent remote controller installed with the application terminal; The device sends an instruction to the smart remote controller, the smart remote controller is in communication with the blood glucose data processing unit, and the blood glucose data processing unit outputs a current corresponding to the voltage according to the instruction; the wireless power supply module is configured according to the input current Voltage value output corresponding to transmit power a sinusoidal signal; the optical module receives a sinusoidal signal to generate an induced current to adjust the luminance of the LED, thereby inducing the light-responsive engineered cells in the graft carrier to express and secrete different amounts of hypoglycemic drugs (specific The optical module includes an inductive receiving coil, a capacitor, an LED, and a graft carrier containing the engineered cells arranged in series; the inductive receiving coil receives an sinusoidal signal to generate an induced current to adjust the luminance of the LED, and induces Engineered cells (eg, photoresponsive engineered cells) in a transplant vector produce varying amounts of hypoglycemic agents).

In the diabetes ultra-long-range intelligent diagnosis and treatment system proposed by the present invention, the blood glucose concentration tester comprises: a blood glucose concentration sensor module, a Bluetooth wireless transmission module (such as a mobile phone Bluetooth wireless transmission module) and a client software thereof. The blood glucose concentration tester may also include a blood glucose test strip. The blood glucose concentration sensor module is a communication device that can convert blood glucose values into electrical signals or digital signals.

In the diabetes ultra-long-range intelligent diagnosis and treatment system according to the present invention, the blood glucose data processing unit determines a blood glucose concentration range, controls different relay switches in the relay unit according to the set blood glucose concentration threshold value, and controls the system through the relay. The output voltage of the switching power supply is used to output a corresponding current to the wireless power supply module.

In the diabetes ultra-long-range intelligent diagnosis and treatment system according to the present invention, the blood glucose data automatic control system further comprises a DC power supply module; a liquid crystal display module for displaying a blood glucose concentration value, an output voltage value, an optical module LED brightness value; Power supply; wireless power supply module.

In the diabetes ultra-long-range intelligent diagnosis and treatment system proposed by the present invention, the blood glucose concentration detector transmits blood glucose concentration data to the blood glucose data processing unit through Bluetooth wireless transmission.

In the diabetes ultra-long-range intelligent diagnosis and treatment system according to the present invention, the mobile device in the blood glucose data remote control system has a wireless receiving module, and the mobile device acquires blood glucose concentration data and reads a blood glucose concentration value by using the application terminal; The smart remote controller and the mobile device communicate remotely by wireless signals, or send remote control commands through a local area network WiFi or a 2G/3G/4G network to control the opening or closing of the light source device, and can be adjusted as needed. Light intensity, lighting time or exposure method.

In the diabetes ultra-long-range intelligent diagnosis and treatment system proposed by the present invention, the blood glucose data remote control system is further provided with a microcontroller, a relay driving module, a relay group, a power adapter and a switching power supply.

In the diabetes ultra-long-range intelligent diagnosis and treatment system proposed by the present invention, the power supply module may be any power source capable of illuminating an LED in an optical module, and may include a low-dropout linear voltage regulator chip, an electromagnetic oscillation circuit, a power amplification circuit, and A transmitting circuit or the like (or the wireless power supply module is further provided with a low-dropout linear regulator chip, an electromagnetic oscillating circuit, a power amplifying circuit, and a transmitting circuit).

In the diabetes ultra-long-range intelligent diagnosis and treatment system proposed by the present invention, the LED emission light includes violet light, blue light, green light, red light, near infrared light, and far red light.

In the diabetes ultra-long-range intelligent diagnosis and treatment system according to the present invention, the transplantation carrier contains engineered cells (such as photo-responsive engineered cells), and the engineered cells are customized cells that are regulated by light-induced regulation of genes, including a plurality of prokaryotic cells and eukaryotic cells; or the light-responsive engineered cells are custom-made cells that are regulated by light-induced regulation of genes, including far-red, red, green, blue, and ultraviolet light-induced regulation of gene expression. A variety of prokaryotic and eukaryotic cells.

In the diabetes ultra-long-range intelligent diagnosis and treatment system proposed by the present invention, the form of the transplantation carrier includes a hydrogel, a semipermeable membrane dialysis bag, a hollow fiber tube, and a microcapsule.

The invention also proposes the application of the diabetes ultra-long-range intelligent diagnosis and treatment system in the treatment of diabetes. The diabetes ultra-long-range intelligent diagnosis and treatment system of the invention tests the blood glucose concentration by the blood glucose concentration tester, and the test data can be fed back to the LED of the optical module in the body through the blood glucose data processing unit and the wireless power supply module, according to the set blood glucose concentration threshold (the blood glucose concentration range) Corresponding to the brightness of the LED), the higher the patient's blood glucose concentration, the stronger the LED brightness of the optical module in the body, and the more hypoglycemic drugs secreted by the light-controlled engineered cells, the more obvious the hypoglycemic effect; when the patient's blood glucose concentration returns to normal or partial When low, the system closes the optical module and stops producing hypoglycemic drugs to prevent the patient from getting too low blood sugar. The system of the invention can convert the blood glucose concentration signal into an optical signal or a user mobile phone control command to control the optical module implanted in the body to precisely adjust the expression level of insulin or glucagon-like peptide, thereby achieving automation, precision and individualized treatment. The purpose of diabetes. The invention integrates diagnosis and treatment of diabetes, has ultra-remote control, and has diabetes The disease condition is accurate, the treatment is accurate and rapid, the operation is simple, the degree of intelligence is high, the treatment is good, and the toxic and side effects are not so far. It has great potential application value in the frontier field of artificial intelligence customized cell and disease diagnosis and treatment.

The present invention also proposes for the first time a new far red light regulation gene expression loop control system (far red light gene loop expression control system). In the present invention, the photon energy of the far red light is much lower than that of the blue photon, and the toxic side effect on the cells is much smaller than that of the blue light. The penetration of far red light is much larger than that of blue light. It can penetrate 7-8 cm of skin and muscle tissue. It can realize the target gene expression of target cells transplanted in the peritoneal cavity without traces, and even regulate the expression of specific tissues and organs in the body. gene. Moreover, the far-red light control system of the present invention can be directly activated by far-red light without additional addition of any photographic pigment. The invention proposes a novel far red light regulation gene expression loop control system by adjusting the amount of the processor in the system and the promoter type in the effector, and the invention optimizes the amount of the processor in the system, the invention The promoter in the effector is also optimized. Its background is lower (SEAP expression is only 1-8U / L), the toxicity to cells is smaller, the system expression ratio is higher (about 50 times), the system is more sensitive to far red light, and the future light control system Deep development and clinical application are more beneficial. The far red light regulating gene expression loop control system of the invention can design different target protein expressions, is used for treating various diseases such as diabetes, has great potential application value, and can be widely popularized in clinical application.

Each of the nucleotide sequences or amino acid sequences described in the present invention can be produced by a synthetic method.

The invention provides an artificially designed and synthesized gene loop control system based on far red light regulating transgene expression. The invention provides an eukaryotic expression vector, an engineered cell or an engineered cell transplantation vector for a far red light regulating gene expression loop control system. The invention also provides kits for the above components of the far red light regulating gene expression loop control system. The invention also provides a novel diabetes therapy based on far red light regulation. The invention can rapidly regulate gene expression, and can regulate the gene expression amount, and has the characteristics of high expression fold, high temporal and spatial specificity, strong tissue penetration, and no toxic and side effects. The expression ratio of the existing STING far red light regulation gene expression loop control system is only about 10 times, and the expression ratio of the far red light regulation gene expression loop control system proposed by the present invention is up to 50 times and the expression background is low ( The expression level of SEAP is only 1-8U/L), which has great potential application value.

The far red light regulation gene expression loop control system of the present invention comprises: a photoreceptor that senses a far red light source; a processor that processes the signal transmitted by the photoreceptor; and an effector that responds to the signal transmitted by the processor .

The photoreceptor of the invention comprises bacterial photo-sensitive diguanylate cyclase BphS and c-di-GMP degrading enzyme YhjH; wherein the amino acid sequence of the BphS is as shown in SEQ ID NO. 15, the coding gene sequence of the c-di-GMP degrading enzyme YhjH is Genebank accession number: ANK04038.

In the present invention, the photoreceptor may further include a phytochrome synthase BphO, and the amino acid sequence of the BphO is shown in SEQ ID NO. The photosensitive diguanylate cyclase BphS converts GTP into c-di-GMP under far-red light conditions, and the photosensitive diguanylate cyclase BphS is one of the most critical proteins as a photoreceptor The core components. The photoreceptor photosensitive diguanylate cyclase BphS is fused from amino acids 1-514 of the BphG protein to amino acids 175-343 of the Slr1143 protein, and mutated 587 arginine of the fusion protein to alanine. The acid (R587A) is prepared; wherein the BphG protein may be derived from Rhodobacter sphaeroides or artificially synthesized, and the BphG protein used in the present invention is artificially synthesized; the Slr1143 protein may be derived from the collecting cell. The algae (Synechocystis sp.) can also be artificially synthesized, and the Slr1143 protein used in the present invention is artificially synthesized.

Wherein, the c-di-GMP degrading enzyme YhjH is derived from Escherichia coli (E. coli.), and can also be artificially synthesized, and the nucleotide sequence encoding the gene is Genebank accession number: ANK04038. The YhjH has a function of degrading c-di-GMP to pGpG [Ryu MH. et al., ACS synthetic biology, 2014, 3(11): 802-810]. Among them, the phytochrome synthase BphO is a hemoglobin oxidase present in Rhodobacter sphaeroides, and BphO can also be artificially synthesized. It has the function of synthesizing the phytochrome biliverdin and provides a phytochrome for BphS synthesis of c-di-GMP [Ryu M H. et al., ACS synthetic biology, 2014, 3(11): 802-810]. Wherein, the amino acid sequences of the BphS and BphO are respectively shown in SEQ ID NO. 15 and SEQ ID NO. 16, and the amino acid sequence of the YhjH is Genebank accession number: NP_417982.

In the present invention, the photoreceptor may further include a promoter that expresses a photoreceptor. Wherein, the photoreceptor-expressing promoter may be any promoter capable of expressing a photoreceptor in a mammalian cell, including but not limited to: a) a simian vacuolating virus promoter SV40 having a nucleotide sequence of SEQ ID NO .1; b) EF1α (may be human EF1α (hEF1α)) promoter, its nucleotide sequence Genebank accession number: AY043301; c) PGK (may be mouse-derived PGK (mPGK)) promoter, Its nucleotide sequence Genebank accession number: HZ040569; d) cytomegalovirus early enhancer and chicken β-actin promoter combined promoter (CAG), its nucleotide sequence Genebank accession number: HQ456319; e) giant cells The viral promoter hCMV has the nucleotide sequence Genebank accession number: KY199427.

Wherein the processor comprises a promoter hCMV (cytomegalovirus promoter), the nucleotide sequence thereof Genebank accession number: KY199427; and an immunosignaling molecule that drives expression of an immune signaling molecule that binds to c-di-GMP to form a binary complex and immunosignaling molecules Self-activation, the immune signaling molecule comprises: a natural immune signaling molecule STING, the nucleotide sequence of Genebank accession number: NM_198282, the source of the STING can be a human source or a mouse source, which contains two functional regions, ie, the N-terminus Functional region with 5 transmembrane structures and spherical carboxy terminal (ie C-terminal) functional region CTD. When STING is expressed in mammalian cells, c-di-GMP can bind to the functional region CTD of STING to form a binary complex and activate it. Activated STING recruits TBK1 through the C-terminal domain to activate it. TBK1 phosphorylates IRF3, followed by dimerization of IRF3 into the nucleus. By adjusting the STING amount of the processor in the system, the invention can make the background of the far red light regulating gene expression loop control system lower (the expression level of SEAP is only 5-8 U/L), and the toxicity to cells is smaller. The mass ratio of the processor to the photoreceptor and the effector is (0.5-2): (0.5-40): (0.5-40); preferably, 1: (1-20): (1-20); further Preferably, it is 1:10:10; it can be adjusted according to different needs.

Furthermore, the processor may also be a complex composed of a polypeptide which is a DNA binding domain and a c-di-GMP binding domain, a polypeptide which is a nuclear localization signal NLS, a polypeptide which is a domain, and a polypeptide which is a transcriptional regulatory domain.

Wherein the polypeptide as a DNA binding domain and a c-di-GMP binding domain, which is a protein which binds to a specific DNA sequence after binding to c-di-GMP, includes a BldD protein, and the amino acid sequence thereof is SEQ ID No.18;

Wherein, the polypeptide as the nuclear localization signal NLS, which may be in the form of 1-3 copies, has an amino acid sequence as shown in SEQ ID NO.

Wherein, the polypeptide as a linking domain may be in a variety of forms from 0 to 30 amino acids in length, and the amino acid sequence thereof is represented by SEQ ID NO. 20 (linked amino acid sequence of a functional peptide (Linker));

Wherein the polypeptide as a transcriptional regulatory domain is a domain protein having a transcriptional activation function.

Wherein the polypeptide as a transcriptional regulatory domain is placed at the N-terminus or C-terminus of the polypeptide BldD of the DNA-binding domain and the c-di-GMP binding domain.

Wherein the effector comprises a promoter P _FRL and a gene gene reporter (nucleic acid sequence of a protein to be transcribed), denoted as P _FRL -reporter.

Wherein the promoter P _FRL comprises a DNA sequence recognized and bound by the dimerized IFR3 and a weak promoter sequence that initiates gene expression. The dimerized IFR3 recognizes and binds to a DNA sequence which is a DNA sequence specifically recognized and bound by the IFR3 polypeptide and which is a partial sequence of the hIFN-RE-ISRE promoter region. Wherein the hIFN-RE-ISRE consists of the nucleotide sequence of the synthetic human interferon response element hIFN-RE as shown in SEQ ID NO. 4 and the interferon-stimulated response element ISRE as shown in SEQ ID NO. . A partial sequence of the hIFN-RE-ISRE promoter region, which is 1-10 copies.

Wherein, the weak promoter for initiating gene expression may be any weak promoter, such as a TATA box having a nucleotide sequence as shown in SEQ ID NO. 2, such as a nucleotide sequence as shown in SEQ ID NO. The cytomegalovirus minimal promoter hCMVmin, and its mutant hCMVmin 3G, etc., do not express or hardly express the downstream gene of interest (the nucleotide sequence to be transcribed) in the absence of the upstream processor. The present invention optimizes the DNA sequence recognized and bound by IFR3 in the effector and the weak promoter, and the DNA sequence recognized and bound by the IFR3 and the weak promoter may be selected from the nucleotide sequence shown in SEQ ID NO. _FRL1 (5×ISRE-h_CMVmin), P _FRL2 (hIFN-RE-h_CMVmin) as shown in SEQ ID NO. 7, and P _FRL3 ((hIFN-RE)-3×ISRE- as shown in SEQ ID NO. h_CMVmin), such as _{P FRL4 ((hIFN-RE)} -3 × ISRE-h_min) shown in SEQ ID NO.9, SEQ ID NO.10 shown as _{P FRL5 ((hIFN-RE)} -3 × ISRE- (hIFN-RE)-3×ISRE-h_min), P _FRL6 ((hIFN-RE)-3×ISRE-h_min-40 bp) as shown in SEQ ID NO. 11, and P as shown in SEQ ID NO. _FRL7 ((hIFN-RE)-h_min) nucleotide sequence, P _FRL8 ((hIFN-RE)-3×ISRE-(hIFN-RE)-h_min) as shown in SEQ ID NO. 13, as SEQ ID NO P _FRL9 (3 x ISRE-(hIFN-RE)-h_min) shown in .14 can make the expression ratio of the system higher. The present invention also optimizes that when the interval between the weak promoter for initiating gene expression and the gene start codon ATG is 40 bp, the gene expression fold is high (about 50 times), and the system background is low (SEAP expression). The quantity is only 5-8U/L), which makes the system more sensitive to far red light, which is more beneficial to the deep development and clinical application of the light control system.

Furthermore, the promoter P _FRL may also be composed of a DNA sequence recognized and bound by the BldD protein and a weak promoter that initiates gene expression, wherein the BldD protein binds to a DNA sequence which is a DNA binding domain and c-di‐ The DNA sequence specifically recognized and bound by the polypeptide of the GMP binding domain is a partial sequence of the bldM promoter region, and the nucleotide sequence is selected from the group consisting of SEQ ID NO. 21 (BldD binding site (bldM) nucleotide sequence), and a partial sequence of the whiG promoter region, the nucleotide sequence is selected from the group consisting of SEQ ID NO. 22 (BldD binding site (whiG) nucleotide sequence), and different combinations of bldM and whiG, different combinations of bldM and whiG, etc. The weak promoter for promoter gene expression includes all weak promoters including the TATA box, the cytomegalovirus hCMV minimal promoter and its mutant hCMVmin 3G.

The present invention optimizes the DNA sequence recognized and bound by BldD in the effector and a weak promoter for inducing gene expression, and the DNA sequence recognized by BldD and the weak promoter for initiating gene expression may be selected from nucleotide sequences such as SEQ. P _{FRL2.1 (1×bldM-h-CMVmin)} represented by ID NO. ₂₃ , P _{FRL2.2 (2×bldM-h-CMVmin)} as shown in SEQ ID NO. 24, as SEQ ID NO. P _{FRL2.3 (3×bldM-h-CMVmin)} shown, P _{FRL2.4 (4×bldM-h-CMVmin)} as shown in SEQ ID NO. 26, P as shown in SEQ ID NO. _{FRL2.5 (5 × bldM-h-} CMVmin), such as _{P FRL2.6 (1 × whiG-h} -CMVmin) shown in SEQ ID NO.28, such as _P FRL2.7 _(shown in SEQ ID NO.29 _{2×whiG-h-CMVmin)} nucleotide sequence, P _{FRL2.8 (3×whiG-h-CMVmin)} as shown in SEQ ID NO. 30, P _FRL2.9 as shown in SEQ ID NO. _{4×whiG-h-CMVmin)} , P _{FRL 2.10 (5×whiG-h-CMVmin)} as shown in SEQ ID NO. ₃₂ , P _FRL2.11 as shown in SEQ ID NO. _{33 (SV40 PolyA-1) × whiG-h-CMVmin)} , P _{FRL2.12 (SV40 PolyA-2×whiG-h-CMVmin)} as shown in SEQ ID NO. 34, P _FRL2.13 as shown in SEQ ID NO. _{35 (SV40 PolyA) -3 × whiG-h-CMVmin)} , _P FRL2.14 as shown in SEQ ID NO.36 _(SV40 _{PolyA-4 whiG-h-CMVmin),} _P FRL2.15 as shown in _{SEQ ID NO.37 (SV40 PolyA-5} × whiG-h-CMVmin), _P FRL2.16 as shown in _SEQ ID NO.38 _{(1 ×} whiG _-h-CMVmin3G) nucleotide sequence, P _{FRL2.17 (2 x whiG-h-CMVmin3G)} as shown in SEQ ID NO. 39, P _FRL2.18 as shown in SEQ ID NO. 40 _{(3 x whiG -h-CMVmin3G)} , P _{FRL 2.19 (4 x whiG-h-CMVmin3G)} as shown in SEQ ID NO. 41, P _FRL 2.20 as shown in SEQ ID NO. _{42 (5 x whiG-h-CMVmin3G) )} , a version with a higher gene expression fold (about 60 times) and better insulation is obtained.

Wherein, the protein encoded by the target gene reporter (nucleotide sequence to be transcribed) may be any protein of interest, including a protein as a reporter gene and/or a pharmaceutical protein or small peptide as a therapeutic disease; Proteins as reporter genes include secreted alkaline phosphatase (SEAP), enhanced green fluorescent protein (EGFP), and luciferase (Luciferase); as a therapeutic protein or small peptide including insulin (Insulin), pancreatic hyperglycemia Prime peptide (GLP-1). Wherein the nucleotide sequence encoding the SEAP Genebank accession number: AX036887, the nucleotide sequence encoding the EGFP Genebank accession number: KY002200, the nucleotide sequence encoding the Luciferase Genebank accession number: KJ561464, encoding the The nucleotide sequence of GLP1-Fc is shown in SEQ ID NO. That is, by adjusting the type of the target gene, the treatment of various diseases or the expression of the target protein can be achieved.

Wherein, when all of the proteins of interest are two or more, they can be simultaneously expressed on an expression vector by self-cleaving peptide 2A, such as SEAP-2A-Insulin, EGFP-2A-Insulin, etc.; The amino acid sequence of the self-cleaving peptide 2A is shown in SEQ ID NO. The 2A sequence used therein can be replaced by an internal ribosome entry site sequence IRES.

In the present invention, the type of the target gene in the system can be adjusted according to the type of disease to be treated to prepare a corresponding product. For example, when treating diabetes, the target gene of the effector is insulin and/or glucagon-like peptide. Gene.

The mechanism of action of the far red light regulating gene expression loop control system of the present invention is that when c-di-GMP is produced under light conditions, c-di-GMP binds to STING to mediate activation of the STING-TBK1-IFR3 signaling pathway. The dimerized IFR3 enters the nucleus, or BldD forms a dimer, recognizes the specific sequence in the effector and binds, and begins to transcribe and express the downstream gene. When c-di-GMP is not produced when the light stops, the synthesized c-di-GMP is degraded, STING cannot be activated, or BldD cannot form a dimer, and the transcriptional expression of the gene cannot be turned on, thereby realizing the gene switch. Close up.

The three components of the far red light regulation gene expression loop control system provided by the present invention can be constructed in a eukaryotic expression vector by genetic engineering technology, thereby realizing the regulation of transcription and expression of the target gene. The far red light regulating gene expression loop control system provided by the invention can temporally and spatially regulate the expression of a target gene in a eukaryotic host cell by using far-red light irradiation which hardly damages cells or the body, the host The cells may be any type of mammalian cells such as hMSC-TERT, Hana 3A, HEK-293A, HEK-293T and the like.

Wherein, the far red light has an illumination intensity of 0-5 mW/cm ² ; the illumination time is 0-72 h; and the illumination method comprises pulsed illumination, continuous illumination, direct illumination or a projection card with a hollow space to spatially control different positions. Irradiation of the gene expression level of the cells. By controlling the far-red light source to generate different illumination times, different expression levels of the regulatory genes are achieved. The far red light source can generate a device with a far-red light of 600-900 nm wavelength, and can be a 600-900 nm LED, an infrared therapeutic device, a laser lamp, or the like.

The invention also proposes a method for constructing the far red light regulating gene expression loop control system. (1) The present invention optimizes the relationship between STING and photoreceptor and processor quality under the promoter hCMV expression, and finds that the ratio between STING and photoreceptor and processor quality is 1:10:10. . (3) The present invention optimizes the effector, and the effector includes a promoter P _FRL and a gene gene reporter, denoted as P _FRL -reporter. Wherein the promoter P _FRL comprises a DNA sequence recognized and bound by the dimerized IFR3 and a weak promoter sequence that initiates gene expression. The weak promoter for initiating gene expression may be any weak promoter, such as a TATA box having a nucleotide sequence as shown in SEQ ID NO. 2, or a giant cell having a nucleotide sequence as shown in SEQ ID NO. The virus minimal promoter hCMV _min , and its mutant hCMVmin 3G and so on. (4) The present invention optimizes the DNA sequence and promoter recognized and bound by IFR3 in multiple versions of the effector, and the DNA sequence and promoter recognized and bound by the IFR3 may be selected from nucleotide sequences such as SEQ ID NO. _{P FRL1 (5 × ISRE-h_CMVmin} ) in FIG. 6, such as _{SEQ ID P FRL2 (hIFN-RE} -h_CMVmin) shown NO.7, SEQ ID NO.8 as shown in P _{FRL3 ((hIFN-RE) -3×ISRE-h_CMVmin)} , P _{FRL4 ((hIFN-RE)-3×ISRE-h_min)} as shown in SEQ ID NO. 9, P _{FRL5 ((hIFN-RE)} as shown in SEQ ID NO _{. -3 x ISRE-(hIFN-RE)-3 x} _{ISRE-h_min)} , P _{FRL6 ((hIFN-RE)-3 x ISRE-h_min-40 bp)} as shown in SEQ ID NO. 11, as SEQ ID NO. The P _{FRL7 ((hIFN-RE)-h_min)} nucleotide sequence shown in 12, P _{FRL8 ((hIFN-RE)-3×} _{ISRE-(hIFN-RE)-h_min)} as shown in SEQ ID _NO. The F _{FRL9 (3 x ISRE-(hIFN-RE)-h_min)} as shown in SEQ ID NO. 14 can make the expression ratio of the system higher. (5) The present invention also optimizes that the gene expression fold is higher (about 50 times) when the interval between the weak promoter and the gene start codon ATG is 40 bp, and the system background is low (the expression of SEAP is only 5-8U/L) makes the system more sensitive to far red light, which is more beneficial to the deep development and clinical application of the light control system.

The invention also proposes a method for constructing the far red light regulating gene expression loop control system, comprising the following steps:

(1) A complex of a polypeptide which is a DNA binding domain and a c-di-GMP binding domain, a polypeptide which is a nuclear localization signal NLS, a polypeptide which is a domain, and a polypeptide which is a transcriptional regulatory domain is constructed as a processor of the system.

Wherein, the polypeptide as a linking domain may have a length from 0-30 amino acids in multiple forms, and the amino acid sequence thereof is shown in SEQ ID NO.

(2) The present invention optimizes the effector, and the effector includes a promoter P _FRL and a gene gene reporter, denoted as P _FRL -reporter. The promoter P _FRL may also be composed of a BldD protein-binding DNA sequence and a weak promoter, wherein the processor BldD protein binds to a DNA sequence which is a polypeptide specificity of a DNA binding domain and a c-di-GMP binding domain The DNA sequence recognized and ligated is a partial sequence of the bldM promoter region, the nucleotide sequence is selected from SEQ ID NO. 21, and is a partial sequence of the whiG promoter region, and the nucleotide sequence is selected from the sequence SEQ ID NO. And the different combinations thereof; the weak promoters that initiate gene expression include all weak promoters, including TATAbox, the cytomegalovirus CMV minimal promoter and its mutant CMVmin 3G. The present invention optimizes the DNA sequence and weak promoter recognized and bound by BldD in the effector, and the DNA sequence and weak promoter recognized and bound by the BldD can be selected from the nucleotide sequence as shown in SEQ ID NO. _FRL2.1 (1×bldM-h _- CMV _min ), P _FRL2.2 (2×bldM-h _- CMV _min ) as shown in SEQ ID NO _. 24, P _FRL2 as shown in SEQ ID NO _{. 3} (3×bldM-h _- CMV _min ), P _FRL2.4 (4×bldM-h _- CMV _min ) as shown in SEQ ID NO. 26, P _FRL2.5 as shown in SEQ ID NO. 5×bldM-h _- CMV _min ), P _FRL2.6 (1×whiG-h _- CMV _min ) as shown in SEQ ID NO. 28, P _FRL2.7 as shown in SEQ ID NO. ₂₉ (2× whiG-h _- CMV _min ) nucleotide sequence, P _FRL2.8 (3×whiG-h _- CMV _min ) as shown in SEQ ID NO. 30, P _FRL2.9 as shown in SEQ ID NO. 4×whiG-h _- CMV _min ), P _FRL2.10 (5×whiG-h _- CMV _min ) as shown in SEQ ID NO. ₃₂ , P _FRL2.11 as shown in SEQ ID NO. ₃₃ (SV40 PolyA) -1×whiG-h _- CMV _min ), P _FRL2.12 (SV40 PolyA-2×whiG-h _- CMV _min ) as shown in SEQ ID NO. 34, P _FRL2 as shown in SEQ ID _{NO. 13} (SV40 PolyA-3×whiG-h _- CMV _min ), P _FRL2.14 as shown in SEQ ID NO. ₃₆ (S V40 PolyA-4×whiG-h _- CMV _min ), P _FRL2.15 (SV40 PolyA-5×whiG-h _- CMV _min ) as shown in SEQ ID NO. ₃₇ , P as shown in SEQ ID NO. _FRL2.16 (1×whiG-h _- CMV _min3G ) nucleotide sequence, P _FRL2.17 (2×whiG-h _- CMV _min3G ) as shown in SEQ ID NO. ₃₉ , as shown in SEQ ID NO. P _FRL2.18 (3×whiG-h _- CMV _min3G ), P _FRL2.19 (4×whiG-h _- CMV _min3G ) as shown in SEQ ID NO. 41, P as shown in SEQ ID NO. _FRL2.20 (5×whiG-h _- CMV _min3G ) obtained a version with a higher gene expression fold (about 60 times) and better insulation.

The invention also proposes a form of construction of the photoreceptor, comprising:

a) synthetic bacteria photosensitive diguanylate cyclase BphS encoding gene BphS;

b) synthetic bacteria photosensitive diguanylate cyclase BphS encoding gene is linked to the c-di-GMP degrading enzyme YhjH encoding gene by the 2A sequence BphS-2A-YhjH;

c) synthetic bacteria photosensitive diguanylate cyclase BphS encoding gene is linked to the phytochrome synthase BphO encoding gene by 2A sequence Bphs-2A-BphO;

d) Synthetic bacteria Photosensitive diguanylate cyclase BphS encoding gene is linked to the phytochrome synthase BphO encoding gene by 2A sequence, and then linked to the c-di-GMP degrading enzyme YhjH encoding gene by 2A sequence BphS-2A- BphO-2A-YhjH;

Wherein the 2A sequence can be replaced by an internal ribosome entry site sequence IRES;

The phytochrome synthase BphO has a function of synthesizing a phytochrome biliverdin;

The c-di-GMP degrading enzyme YhjH has a function of degrading c-di-GMP to pGpG.

The amino acid sequences of the BphS and BphO are shown in SEQ ID NO. 15 and SEQ ID NO. 16, respectively, and the amino acid sequence accession number of the YhjH is NP_417982.

The invention also provides a eukaryotic expression vector, an engineered cell or an engineered cell transplantation vector containing the far red light regulating gene expression loop control system; wherein the engineered cell transplantation carrier comprises a hollow fiber membrane transplantation tube, Sodium alginate rubber blocks, etc.

The invention also proposes a kit comprising the far red light regulating gene expression loop control system. The invention also proposes a kit comprising a eukaryotic expression vector containing the far red light regulating gene expression loop control system and/or a host cell and/or engineering transfected with the eukaryotic expression vector Cell transplantation vector and corresponding instructions.

In the present invention, the kit includes a plasmid kit for regulating each component of the far red light regulating gene expression loop control system, and a mammalian cell kit containing a control system for regulating the far red light regulating gene expression loop. And the corresponding instructions.

The invention also proposes a method for preparing a eukaryotic expression vector, an engineered cell or an engineered cell transplantation vector containing the far red light regulating gene expression loop control system. The eukaryotic expression vector comprises a mammalian cell expression vector comprising the far red light regulatory gene expression loop control system. The expression vector may be a vector containing a far-red photoreceptor-encoding gene alone or a vector containing a processor-encoding gene alone or a vector containing an effector-encoding gene, and the effector contains a far-red light-responsive promoter. However, it does not contain the nucleic acid sequence to be transcribed. Alternatively, the expression vector comprises two or three of a vector encoding a far red photoreceptor gene, a vector encoding a processor gene, and a vector encoding an effector gene. The construction of all the mammalian cell expression vectors described above is shown in Table 2.

The invention also provides the use of a eukaryotic expression vector comprising the far red light regulating gene expression loop control system for the preparation of a medicament for treating diabetes, comprising type I diabetes and/or type II diabetes.

The invention also proposes the use of the far red light regulating gene expression loop control system for preparing a therapeutic drug for diabetes, the type comprising diabetes type I and/or type II diabetes. The invention provides a safe, reliable and precise strategy for the precise regulation of insulin release and glucagon-like peptides in the treatment of diabetes in time and space. The present invention provides new methods and strategies for treating diabetes. The system can regulate insulin And/or expression of the glucagon-like peptide GLP-1. The expression construct of the insulin includes SEAP-2A-Insulin, EGFP-2A-Insulin, EGFP-2A-SEAP-2A-Insulin. Expression of the glucagon-like peptide GLP-1 includes GLP-1-Fc and the like. The far red light regulating gene expression loop control system of the invention can rapidly regulate gene expression through far red light, accurately control gene expression amount, has high regulation gene expression multiple, high temporal and spatial specificity, strong tissue penetration and non-toxicity Side effects and other characteristics.

DRAWINGS

Figure 1 shows the overall working principle of the diabetes ultra-long-range intelligent diagnosis and treatment system.

Figure 2 is a schematic circuit diagram of a diabetes ultra-long-range intelligent diagnosis and treatment system.

Figure 3 is a blood glucose concentration tester, a Bluetooth wireless transmitter module, and a DC power supply module physical map of the blood glucose data automatic control system.

4 is a physical diagram of a liquid crystal display module of a blood glucose data automatic control system.

Figure 5 is a physical map of the wireless power supply module and the optical module far away from the red LED coil in a certain range.

Figure 6 is a physical diagram of the intelligent controller of the blood glucose data remote control system.

Figure 7 is a screenshot of the APP of the intelligent controller of the blood glucose data remote control system.

Figure 8 is a physical diagram of the optical module.

Fig. 9 is a schematic view showing the structure of an optical module.

Figure 10 is a physical diagram of the inductive receiving coil, capacitor, and far-infrared LED of the optical module.

Figure 11 is a graph showing experimental results in vitro to verify that different concentrations of glucose solution can control the different brightness of the in-vivo optical module far-red LEDs to induce different SEAP expression levels.

Figure 12 is a graph showing the results of an experiment in which blood samples of different blood glucose concentrations were taken from wild-type mice and diabetic mice in vitro to control the different brightness of the in-vivo optical module far-red LEDs to induce different insulin expression levels in mice.

Figure 13 is a graph showing experimental results of in vitro validation of blood samples from different blood glucose concentrations in healthy and diabetic patients to control the different brightness of the in vivo optical module far-red LEDs to induce different GLP-1-Fc expression levels.

Figure 14 is a graph showing experimental results of a digital self-reward function for different blood glucose concentrations in a wild-type mouse and a type II diabetic mouse using SEAP as a reporter gene in wild-type mice and type II diabetic mice.

Figures 15, 16, and 17 are graphs showing the results of diagnosis and treatment of type 2 diabetes mellitus in a diabetic ultra-long-range intelligent diagnosis and treatment system in type 2 diabetic mice.

Among them, Fig. 15 is a graph showing the expression levels of GLP-1-Fc induced by the diagnosis and treatment of type 2 diabetic mice at 24h, 48h, and 72h by the diabetes ultra-long-range intelligent diagnosis and treatment system.

Among them, Figure 16 is a graph showing the results of blood glucose concentration measured at 24h, 48h, and 72h in the diagnosis and treatment of type 2 diabetic mice by the diabetes ultra-long-range intelligent diagnosis and treatment system.

Among them, Fig. 17 is a diagnosis and treatment of type II diabetic mice by the diabetes ultra-long-range intelligent diagnosis and treatment system, and the brightness of the optical module far-red LED and the corresponding blood glucose concentration result at the time of each blood glucose monitoring.

18 is a schematic diagram showing the principle of a STING-based far red light regulation gene expression loop control system in mammalian cells.

19 is a schematic diagram showing the principle of a BldD-based far red light regulation gene expression loop control system in mammalian cells.

Figure 20 is a graph showing experimental results of photoreceptors expressed by different promoters of the far red light regulating gene expression loop control system of the present invention.

21 is a graph showing experimental results of different amounts of processors of the STING-based far red light regulation gene expression loop control system of the present invention.

22 is a diagram showing experimental results of different configurations of a processor based on BldD-based far red light regulation gene expression loop control system.

Figure 23 is a graph showing experimental results of different effectors constructed by the STING-based far red light-regulated gene expression loop control system of the present invention.

24-27 are diagrams showing experimental results of effectors constructed by BldD-based far-red light-regulated gene expression loop control system according to the present invention.

Figure 28 is a graph showing the results of experimental expression of a far red light-regulated gene expression loop control system in different mammalian cells.

Figure 29 is a graph showing experimental results of different expression levels of different illumination time-regulated far-red light-regulated gene expression loop control systems of the present invention.

Figure 30 is a graph showing the effect of different light intensities on the expression of a target protein in a far red light-regulated gene expression loop.

Figure 31 is a graph showing the effect of different illumination times on the expression level of the target protein FLuc activity of the far red light-regulated gene expression loop control system of the present invention.

Figure 32 is a diagram showing the expression of the control system of the far red light regulating gene expression loop in different illumination time according to the present invention The protein has an effect on the amount of active GLP-1 expression.

Figure 33 is a diagram showing the results of green fluorescence experiments in which two far-end light-regulated gene expression loop control systems can simultaneously express two or more proteins of interest.

Figure 34 is a diagram showing the results of an experimental experiment of insulin in two or more of all significant proteins simultaneously expressing the far-red light-regulated gene expression loop control system of the present invention.

Fig. 35 is a view showing the results of an experiment for preparing a hollow fiber membrane graft graft carrier containing engineered cells of a far red light regulating gene expression loop control system according to the present invention.

Figure 36 is a graph showing the results of the far red light toxicity test of the present invention.

Figure 37 is a background measurement result of the far red light regulating gene expression loop control system of the present invention.

Fig. 38 is a diagram showing the results of an experiment in which a far red light-regulated gene expression loop control system is regulated by far-red light in a mouse.

Figure 39 is a diagram showing the fasting blood glucose level of the far-red light-regulated gene expression loop control system of the present invention for accurately regulating insulin expression in type I diabetes in a type I diabetes model mouse.

40 is a result of the glucose tolerance test of the far-red light regulating gene expression loop control system of the present invention for accurately regulating insulin expression in type I diabetes in a type I diabetes model mouse.

Figure 41 is a diagram showing the fasting blood glucose level of the far-red light-regulated gene expression loop control system of the present invention for accurately regulating GLP-1-Fc expression in type II diabetes mice.

Figure 42 is a graph showing the results of the glucose tolerance test of the far-red light-regulated gene expression loop control system of the present invention for accurately regulating GLP-1-Fc expression in type II diabetes mellitus in the treatment of type 2 diabetes mellitus.

Figure 43 is a graph showing the results of insulin resistance test of type 2 diabetes mellitus in the type II diabetes mellitus model in the type II diabetes model of the present invention.

Figure 44 is a graph showing the amount of glucagon expressed by type II diabetes in the type II diabetes mellitus in a type II diabetes model mouse controlled by a far red light regulating gene expression loop control system of the present invention.

detailed description

The present invention will be further described in detail in conjunction with the following specific embodiments and drawings. These examples are for illustrative purposes only and are not to be construed as limiting the scope of the invention. The processes, conditions, experimental methods, and the like of the present invention are generally known in the art and common general knowledge, except for the contents specifically mentioned below, and the present invention is not particularly limited. The reagents, instruments, and the like used in the following examples, as well as the experimental methods not specifying the specific conditions, are carried out according to the conditions recommended by the conventional or commercial suppliers.

First, diabetes ultra-long-range intelligent diagnosis and treatment system

The diabetes ultra-long-range intelligent diagnosis and treatment system of the invention comprises a blood sugar data automatic control system, a blood glucose data remote control system, a wireless power supply module and an optical module. The blood sugar data automatic control system includes a blood glucose concentration detector and a blood glucose data processing unit; the blood glucose concentration detector in the blood glucose data automatic control system acquires the blood glucose concentration value, and generates blood glucose concentration data; and the blood glucose data processing unit in the automatic blood sugar data control system Extracting a blood glucose concentration value from the blood glucose concentration data, and outputting a current corresponding to the voltage according to the blood glucose concentration value to the wireless power supply module;

The blood glucose data remote control system includes a mobile device and an intelligent remote controller installed with the application terminal; the two are connected to the same wireless local area network WLAN, and the mobile device sends an instruction to the intelligent remote controller, and inputs the blood glucose data through the intelligent remote controller. Outputting a current corresponding to the voltage in the unit;

The wireless power supply module outputs a sine wave signal corresponding to the transmit power according to the voltage value of the input current; the optical module includes an inductive receiving coil, a capacitor, an LED, and a hydrogel containing the engineered cells arranged in series; the inductive receiving coil receives the sine wave signal An induced current is generated to adjust the illuminance of the optical module LEDs, inducing engineered cells in the hydrogel to produce different amounts of hypoglycemic agents. At the same time, the brightness of the LED of the optical module is remotely adjusted, and the engineered cells in the hydrogel are induced to produce different amounts of hypoglycemic drugs.

The blood glucose concentration tester used in the specific embodiment is purchased from Beijing Yicheng Bioelectronics Technology Co., Ltd., and is modified, and the data is docked with the mobile phone Bluetooth wireless transmitting and receiving module to complete the wireless transmission of blood glucose concentration data. The transmission test distance is within 10m. The blood glucose concentration detector uses a 3.7V polymer rechargeable lithium battery to power the blood glucose reading instrument and the Bluetooth transmitter module.

The blood glucose data automatic control system in the specific embodiment further includes a DC power supply module, a blood glucose data processing unit, a liquid crystal display module, a relay unit, and a switching power supply. The DC power supply module is an AC-DC power adapter, which converts the AC power of the AC220V, and converts the DC voltage to the switching power supply module, the blood glucose data processing unit, the Bluetooth wireless transmitting and receiving module, the liquid crystal display module, and the relay unit. The blood glucose data processing unit is a low-power series blood glucose data processing unit chip MSP430 of Texas Instruments, which is connected with the Bluetooth wireless transmitting and receiving module, and receives the blood glucose concentration data transmitted by the Bluetooth wireless transmitting module through a standard universal serial asynchronous transceiver bus. The blood glucose data processing unit MSP430 extracts the received blood glucose data according to the communication protocol, and transmits the data to the display module for display, and simultaneously determines the blood glucose concentration range, and controls different relay switches according to the threshold division of the concentration, thereby regulating the The system switches the output voltage of the power supply to achieve brightness control of the optical module in the body.

Blood glucose concentration threshold is divided into <6.1mM, 6.1-11.1mM, 11.1-16.8mM,> 16.8mM, far-red light respectively corresponding to the brightness of the LED is ^{0mW / cm 2, 0.2mW / cm} 2, 1.0mW / cm 2, 5.0mW/cm ² .

The following is the design of the blood glucose concentration control program:

The above blood glucose concentration threshold and its pseudo code are one of the specific embodiments of the present invention, and the present invention does not limit the above threshold.

The blood glucose data remote control system in the specific embodiment includes: a mobile device installed with an application terminal, a smart remote controller, a micro controller, a relay drive module, a relay group, a power adapter, and a switching power supply.

In the above specific implementation manner, the input voltage of the switching power supply is 5 to 23V, the highest is 23V, and the use is excellent within 20V, and the input is anti-reverse (the input voltage must be higher than the output voltage by more than 1V); the adjustable output voltage range 0V ~ 16.5V continuously adjustable, automatically save the last set voltage; peak current 3A, used in 2A is excellent. Accuracy 1%, minimum display 0.01A; high conversion efficiency, up to 95% (efficiency related to input, output voltage, current, voltage difference); load regulation S (I) ≤ 0.8%, voltage adjustment rate S (u) ≤0.8%. The mobile device is one or more smart phones, and the application terminal is a corresponding client software App. The intelligent remote controller supports mobile phone remote control, supports WiFi, 2G/3G/4G control mode, allows up to 10A high current, supports multi-channel control, each channel can be independently controlled, and the control effect is pushed to the mobile device in real time, and is in the application terminal. Display; intelligent remote controller can also support time switch, scene mode, realize time switch and one-button switch function, support Android, Apple mobile phone and tablet, software supports custom attributes, the same software supports multiple devices, multiple switches, It can further support data backup and data recovery, support scanning QR code import device, and share equipment with other personnel, and the operator is convenient.

The wireless power supply module includes: a low dropout linear regulator chip, an electromagnetic oscillating circuit, a power amplifying circuit, and a transmitting circuit. The low-dropout linear regulator chip provides the stable voltage required by the module. The electromagnetic oscillation circuit generates a sine wave signal with a frequency of 180KHz. The power amplifier circuit provides the power required for the sine wave signal with a frequency of 180KHz, and the transmitting circuit transmits the sine wave signal to Space creates an electromagnetic environment. Among them, the transmitting circuit is made of a pure copper wire and has a transmitting coil with an outer diameter of 20 cm and a plurality of transmitting coils to form a relatively uniform wireless electromagnetic environment.

The optical module includes an inductive receiving coil, a capacitor, an LED, and a hydrogel containing engineered cells. The LEDs are two red-emitting LEDs in a 3535 package. Further, the hydrogel LED encapsulates the engineered mammalian cell HEK-293 regulated by far red light, and the preparation process is as follows: HEK-293 cells transfected with the far red light system are suspended at 1.5% ( w/v) in sodium alginate buffer (sodium alginate is dissolved in DMEM), reaching a final cell density of 4 × 10 ⁶ cells per ml, equivalent to 2 × 10 wrapped in each hydrogel LED ^Six HEK-293 cells transfected as described above. The far-red LED coil of the optical module was placed face up on the bottom of the 24-well plate hole, and 500 μl of the above cell suspension droplets were pipetted onto the far-red LED coil. Then, 500 μl of polymerization buffer (100 mM CaCl ₂ , 10 mM MOPS, pH=7.2) was added to cure for more than 10 min, and finally placed in a 0.05% poly-lysine solution (0.05% poly-L-lysine, MW: 15,000-30,000, Incubate for 10 min in 10 mM MOPS, 0.85% NaCl, pH = 7.2).

Molecular Cloning

Molecular Cloning Techniques All of the expression plasmids of the present invention are constructed, and the steps are common knowledge in the art.

All primers used for PCR were synthesized by Jinweizhi Biotechnology Co., Ltd. The expression plasmids constructed in the examples of the present invention were all sequenced, and the sequence determination was performed by Jinweizhi Biotechnology Co., Ltd. The Phanta Max Super-Fidelity DNA polymerase used in the examples of the present invention was purchased from Nanjing Nuoweizan Biotechnology Co., Ltd. Endonucleases and T4 DNA ligase were purchased from TaKaRa. The homologous recombinase was purchased from Heyuan Biotechnology (Shanghai) Co., Ltd. Phanta Max Super-Fidelity DNA Polymerase is purchased with the corresponding polymerase buffer and dNTP. Endonucleases, T4 DNA ligase, and homologous recombinases are purchased with the corresponding buffer. Yeast Extract, Trypton, Agar Powder, and Ampicillin (Amp) were purchased from Shanghai Shenggong Bioengineering Technology Co., Ltd. DNA Marker DL5000, DNA Marker DL2000 (Bao Bioengineering Co., Ltd.); Nucleic Acid Dye EB (Guangdong Guoao Biotechnology Co., Ltd.); Plasmid Small Extraction Kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.); DNA Glue Recovery Kit The PCR product purification kits are all purchased from Kangwei Century Biotechnology Co., Ltd.; the remaining reagents such as anhydrous ethanol and NaCl mentioned in the examples are domestic analytical pure products. Glue recovery, purification and recovery of DNA fragments, the steps are based on the DNA gel recovery kit, PCR product purification kit (Kang Wei Century Biotechnology Co., Ltd.) operating instructions; plasmid extraction step based on plasmid extraction (Tiangen Biochemical Technology (Beijing) ))) Extraction kit instructions.

Cell culture and transfection

In the examples of the present invention, the following cell lines and PEI transfection are used as an example to illustrate the operation of the gene loop remote control system of far red light regulating transgene expression in cells and animals, but the scope of the present invention is not limited.

10 cm cell culture dishes for cell culture, cell culture plates (24 wells), 15 mL and 50 mL centrifuge tubes were purchased from Thermo Fisher Scientific (Labserv), USA; modified Eagle medium, fetal bovine serum, penicillin used. The streptomycin solution was purchased from Gibico, USA; the PEI used for transfection was purchased from Polysciences; the cell culture incubator was purchased from Thermo Fisher Scientific, USA; the remaining consumables were common domestic consumables.

Cell culture: Human embryonic kidney cells (HEK-293, ATCC: CRL-11268) were cultured in modified Eagle medium supplemented with 10% (v/v) fetal bovine serum and 1% (v/v). Penicillin and streptomycin solution; the cells were cultured in an incubator containing 5% carbon dioxide at 37 °C.

Transfection: All cell lines were transfected using the optimized PEI procedure (Wieland M, Methods 56(3): 351). Briefly, 4×10 ⁶ cells were seeded in a 10 mL 10 cm cell culture dish, and after 16 h of culture, the optimal ratio of DNA was mixed with PEI in a mass ratio of 3:1 (PEI:DNA). Dissolved in 2 mL of medium and allowed to stand for 6 h.

Detection of reporter secreted alkaline phosphatase (SEAP)

High arginine, magnesium chloride, diethanolamine and HCl for the detection of the reporter gene reaction buffer were purchased from Bioengineering (Shanghai) Co., Ltd.; chromogenic substrate (pNPP: p-Nitrophenylphosphate) was purchased from Shanghai Jingjing Pure Biotechnology Co., Ltd. (Aladdin).

(1) Reagent configuration:

2x buffer:

· 20 mM high arginine Note: its role is to inhibit endogenous alkaline phosphatase activity

·1 mM magnesium chloride

·2% diethanolamine

· Adjust pH to 9.8 with HCl

Substrate solution:

· 120 mM chromogenic substrate (pNPP: p-Nitrophenylphosphate)

·In 2x assay buffer

(2) Experimental steps:

1. Pipette the cell culture supernatant, 200 μL into the centrifuge tube (Note: generally exceed 150 μL, because subsequent heating will lose a part of the volume).

2. 65 ° C water bath for 30 min (Note: heating is mainly to let endogenous alkaline phosphatase inactivated, while SEAP is resistant to high temperatures, will not be inactivated at this temperature).

3. Pipette 80 μL (diluted according to the experimental conditions) into a 96-well plate, and quickly add 20 μl of 2x buffer and 20 μl of the substrate solution pre-warmed.

4. The microplate reader was measured 10 times at 405 nm, with an interval of 1 min each time (conditions can be set according to the experimental conditions).

(3) Calculation of enzyme activity

The enzyme activity of alkaline phosphatase (SEAP) is defined as: the reaction of the substrate p-nitrophenyl phosphate disodium (PNPP-Na ₂ ) to 1 mol/L p-nitrophenol in 1 min at 37 ° C, pH 9.8. A phosphatase, defined as 1 vitality unit (1 U). The p-nitrophenol itself has a bright yellow color, and at a wavelength of 405 nm, different concentrations of p-nitrophenol correspond to different absorbance values. The calculation method is as follows: the OD value measured at different time points in the reaction process of the sample and the substrate is the slope of the curve *256.8 is the enzyme activity, the unit U/L.

Preparation of optical module hydrogels The sodium alginate used in far-red LEDs was purchased from Buchi, Switzerland. Polylysine was purchased from Sigma, USA. Sodium chloride, calcium chloride and MOPS (Morpholinopropanesulfonic acid) were purchased from Shanghai Biotech. Bioengineering Technology Co., Ltd.

Insulin detection method

The Mouse Insulin ELISA kit used in the experiment was purchased from Mercodia, Sweden. The specific measurement method is detailed in the product manual.

Method for detecting glucagon (GLP-1-Fc)

The glucagon detection kit (Millipore Corporation, Billerica, MA 01821USA, Cat. no. EGLP-35K, Lot. no. 2639195) used in the experiment was purchased from Millipore Corporation of the United States, and the specific measurement method is detailed in the product specification.

Embodiment 1, the composition and production of a diabetes ultra-long-range intelligent diagnosis and treatment system

In the present invention, FIG. 2 is taken as an example to illustrate a method for manufacturing a diabetes ultra-long-range intelligent diagnosis and treatment system, but does not limit the scope of protection of the present invention.

In the first step, the blood glucose meter was modified to incorporate a Bluetooth wireless transmitter module and a 3.7V polymer rechargeable lithium battery. Bluetooth serial port transmitting and receiving module model is HC-05, master-slave integrated Bluetooth module, integrated universal serial asynchronous transceiver bus, interface level is international standard TTL level, HC-05 master-slave integrated Bluetooth module and blood glucose The concentration tester performs data docking to wirelessly transmit blood glucose concentration data, and the test distance is 10m; the 3.7V polymer rechargeable lithium battery supplies power to the blood glucose concentration tester and the Bluetooth transmitting module.

In the second step, the blood glucose data processing unit and the Bluetooth wireless receiving module are connected. The blood glucose data processing unit, that is, the microcontroller (MCU), is a low-power series microcontroller chip MSP430 of Texas Instruments, and is connected with a Bluetooth wireless receiving module, and receives the Bluetooth transmitting module transmission through a standard universal serial asynchronous transceiver bus. Blood glucose concentration data. The blood glucose concentration tester of the blood glucose data automatic control system, the Bluetooth wireless transmitting and receiving module, and the DC power supply module physical map are shown in Figure 3.

In the third step, the blood glucose data processing unit and the liquid crystal display module are connected. The microcontroller MSP 430 extracts the received blood glucose data according to the communication protocol, and transmits the data to the liquid crystal display module for display. The blood glucose data automatic control system liquid crystal display module physical map is shown in Figure 4 of the manual.

In the fourth step, the blood glucose data processing unit is connected to the relays in different modes. The blood glucose data processing unit determines the blood glucose concentration range, and outputs corresponding to different concentration ranges according to the threshold value of the blood glucose concentration. The high and low level signals drive the relay switch states in different modes.

In the fifth step, the relay and the wireless power supply module are connected, and the wireless power supply module and the optical module are in a certain range. The state of the relay switch in different modes determines the transmit power of the wireless power supply module, thereby regulating the output voltage of the system switching power supply to achieve brightness adjustment of the optical module. The physical diagram of the wireless power supply module and the optical module far away from the red LED coil in a certain range is shown in Figure 5 of the specification.

The sixth step is the implementation of the App client. The matching App provided by the purchased intelligent controller manufacturer (see the experimental materials and methods), the specific settings and how to use them are detailed in the manufacturer's instruction manual. The function of the app includes controlling the brightness, illumination time and timing switch of the far-red LED of the optical module; also displaying the blood glucose concentration value of the current patient body and the brightness value of the far red LED; and recording the blood sugar condition of the patient , the App client sent to the mobile device held by its guardian. The blood glucose data remote control system intelligent controller supporting App screenshot is detailed in Figure 7 of the manual.

The seventh step is the realization of the remote control system of blood glucose data. The blood glucose data remote control system (specific functions and parameters are detailed in experimental methods and materials) of the present invention is purchased from a smart home studio, through which a smart phone can be used to realize direct ultra-remote control using LAN WiFi or 2G/3G/4G network resources. The brightness of the far red light source controls the amount of expression of different SEAP, insulin or GLP-1-Fc. The blood glucose data remote control system intelligent controller physical map is shown in Figure 6 of the manual.

The eighth step is the production of optical modules. The optical module of the present invention consists of an inductive receiving coil, a capacitor, two patched 3535-packed far-infrared LEDs, and a hydrogel encased with engineered cells that are controlled by far-red light. The capacitor and the far-infrared LED are in a parallel relationship. Engineered cells that are regulated by far red light are HEK-293 cells that are required for transfection. The physical diagram of the optical module is shown in Figure 8 of the specification. The structure mode diagram is shown in Figure 9 of the specification. The physical diagram of the induction receiving coil, capacitor and far-infrared LED is shown in Figure 10 of the specification.

Example 2, in vitro verification of different concentrations of glucose solution through the diabetes ultra-long-range intelligent diagnosis and treatment system can control the different brightness of the optical module far red LED to induce different SEAP expression levels of the engineered cells in the hydrogel

In this example, SEAP is used as a reporter gene, and it is exemplified that different concentrations of the glucose solution can control different brightness of the in-vivo optical module far-red LED, thereby inducing different SEAP expression levels, but there is no limitation on the scope of protection of the present invention. Specific steps are as follows:

The first step is the production of diabetes ultra-long-range intelligent diagnosis and treatment system (see the specific implementation materials and Method and embodiment 1)

The second step is plasmid construction. The plasmid construction in this example is shown in Table 1.

In the third step, the cells are inoculated. HEK-293 cells in good growth state were digested with 0.25% trypsin and seeded in 10 cm cell culture dishes, 4 × 10 ⁶ cells per dish, and 10 mL of DMEM medium containing 10% FBS was added.

The fourth step is transfection. After inoculation of the cells for 16 to 24 hours, 4 μg of pWS46, 4 μg of pGY32, 4 μg of pXY34, PEI transfection reagent and serum-free DMEM were mixed, allowed to stand at room temperature for 15 min, and then uniformly added dropwise to a 10 cm cell culture dish. The total volume of each dish was 2 mL, and the mass ratio of plasmid to PEI was 1:3. After 6 h of transfection, 10 mL of DMEM medium containing 10% FBS was exchanged for culture.

The fifth step is the preparation of a hydrogel far-red LED (refer to the materials and methods for specific methods).

In the sixth step, the hydrogel far-red LED prepared above is placed in a 24-well plate for cultivation, and the glucose solutions with different glucose concentration thresholds are respectively prepared (concentrations are 2 mM, 4 mM, 6 mM, 7 mM, 9 mM, respectively). 11 mM, 12 mM, 14 mM, 16 mM, 18 mM, 20 mM, 22 mM total 12 groups) were respectively added to the blood glucose test strip, and the blood glucose concentration detector obtained the blood glucose concentration value, and the blood glucose concentration data was generated; The blood glucose data processing unit extracts a blood glucose concentration value from the blood glucose concentration data, and outputs a current corresponding to the voltage according to the blood glucose concentration value to the wireless power supply module; the wireless power supply module outputs a sine wave signal corresponding to the transmission power according to the voltage value of the input current; the optical module includes Inductive receiving coil, capacitor, far red LED and graft carrier containing engineered cells arranged in series; inductive receiving coil receives sinusoidal signal to generate induced current to adjust the brightness of the optical module far red LED, induce hydrogel The above engineered cells produce different amounts of SEAP expression.

The seventh step is to detect the reporter gene. After 48 hours of culture, the cell culture supernatants of each group were taken to determine the expression level of SEAP (specific methods refer to materials and methods).

For example, a glucose solution having a concentration of 2 mM, 4 mM, 6 mM is added dropwise to a blood glucose test strip, the concentration being within a threshold range of <6.1 mM, and the brightness of the corresponding far red LED is 0 mW/cm ² , thus measuring The expression level of SEAP in the supernatant of the 24-well plate was almost 0; the glucose solution with a concentration of 7 mM, 9 mM, 11 mM was added dropwise to the blood glucose test strip, which was within the threshold range of 6.1-11.1 mM, corresponding to the far The brightness of the red LED is 0.2mW/cm ² , so the expression of SEAP in the clear solution of the 24-well plate is about 100U/L; the glucose solution with the concentration of 12mM, 14mM, 16mM is added to the blood glucose test strip. Above, the concentration is in the threshold range of 11.1-16.8 mM, and the brightness of the corresponding far red LED is 1.0 mW/cm ² , so that the expression level of SEAP in the clear liquid in the 24-well plate is about 200 U/L; A glucose solution having a concentration of 18 mM, 20 mM, 22 mM was added dropwise to the blood glucose test strip. The concentration was within a threshold range of >16.8 mM, and the brightness of the corresponding far red LED was 5.0 mW/cm ² , thus 24 holes were measured. The expression level of SEAP in the plate supernatant is about 300 U/L.

The results show that the blood glucose data automatic control system in the ultra-long-range intelligent diagnosis and treatment system of diabetes can output the current of the corresponding voltage according to the glucose solution of different glucose concentration thresholds input to the wireless power supply module, and the induction receiving coil of the optical module receives the sine wave signal and generates The induced current is used to adjust the illuminance of the far-red LED of the optical module, and the above-mentioned engineered cells in the hydrogel are induced to generate different SEAP expression levels. In this embodiment, SEAP is used as a reporter gene to verify the function of the diabetes ultra-long-range intelligent diagnosis and treatment system in vitro. The experimental data is detailed in Figure 11 of the specification.

Example 3, in vitro verification of blood samples taken from wild-type mice and diabetic mice with different blood glucose concentrations. The diabetes ultra-remote intelligent diagnosis and treatment system can control the different brightness of the optical module far-red LEDs to induce engineering in the hydrogel. Cells produce different levels of insulin expression in mice

In this example, mouse insulin is used as a reporter gene, and it is exemplified that different concentrations of blood glucose can control the different brightness of the optical module to induce different insulin expression levels in mice, but the scope of protection of the present invention is not limited. Specific steps are as follows:

The first step is the production of a diabetes ultra-long-range intelligent diagnosis and treatment system (see the specific embodiment materials and methods and Example 1 above).

The fourth step is transfection. After inoculation of the cells for 16 to 24 hours, 4 μg of pWS46, 4 μg of pGY32, 4 μg of pWS213, PEI transfection reagent and serum-free DMEM were mixed, allowed to stand at room temperature for 15 min, and then uniformly added dropwise to a 10 cm cell culture dish. The total volume of each dish was 2 mL, and the mass ratio of plasmid to PEI was 1:3. After 6 h of transfection, 10 mL of DMEM medium containing 10% FBS was exchanged for culture.

In the sixth step, the hydrogel far-red LEDs prepared above were placed in a 24-well plate for culture, and blood samples of different blood glucose concentrations taken from wild-type mice and diabetic mice were respectively 5.7 mM. , 8.4 mM, 14.8 mM, 21.2 mM. Dropping on the blood glucose test strip, the blood glucose concentration detector obtains the blood glucose concentration value, and generates blood glucose concentration data; and the blood glucose data processing unit in the blood glucose data automatic control system extracts the blood glucose concentration value from the blood glucose concentration data, according to the blood glucose concentration value Outputting a current corresponding to the voltage to the wireless power supply module; the wireless power supply module outputs a sine wave signal corresponding to the transmit power according to the voltage value of the input current; the optical module includes an inductive receiving coil, a capacitor, a far red LED, and an engineered cell arranged in series The graft carrier; the inductive receiving coil receives the sinusoidal signal to generate an induced current to adjust the luminance of the optical module far red LED, and induces the above-mentioned engineered cells in the hydrogel to produce different mouse insulin expression levels. The seventh step is to detect the reporter gene. After 48 hours of culture, the cell culture supernatants of each group were used to determine the expression level of insulin in mice (specific methods refer to materials and methods).

For example, a blood sample having a blood glucose concentration of 5.7 mM is dropped onto a blood glucose test strip, the concentration is within a threshold range of <6.1 mM, and the brightness of the corresponding far red LED is 0 mW/cm ² , thus 24 holes are measured. The amount of mouse insulin in the plate supernatant was about 1.5 ng/mL; the blood sample with a blood glucose concentration of 8.4 mM was added to the blood glucose test strip, and the concentration was within the threshold range of 6.1-11.1 mM, corresponding to the far red The brightness of the light LED was 0.2 mW/cm ² , so that the expression of mouse insulin in the clear liquid in the 24-well plate was about 4.5 ng/mL; the blood sample with the blood glucose concentration of 14.8 mM was added to the blood glucose test strip. The concentration is in the threshold range of 11.1-16.8 mM, and the brightness of the corresponding far red LED is 1.0 mW/cm ² , so that the expression of mouse insulin in the supernatant of the 24-well plate is about 7.5 ng/mL; A blood sample having a concentration of 21.2 mM was added dropwise to the blood glucose test strip. The concentration was within a threshold range of >16.8 mM, and the brightness of the corresponding far red LED was 5.0 mW/cm ² , thereby measuring the 24-well plate. The amount of mouse insulin expressed in the solution was approximately 12.5 ng/mL.

The results show that the blood glucose data automatic control system in the diabetes ultra-long-range intelligent diagnosis and treatment system can output the corresponding voltage current to the wireless power supply module according to the input blood sample with different blood glucose concentration thresholds (taken from wild type mice and diabetic mice). The inductive receiving coil of the module receives the sine wave signal to generate an induced current to adjust the brightness of the far-red LED of the optical module, and induces the above-mentioned engineered cells in the hydrogel to produce different amounts of insulin expression in the mouse. In this example, mouse insulin was used as a reporter gene to verify the function of the diabetes ultra-long-range intelligent diagnosis and treatment system in vitro. The experimental data is detailed in Figure 12 of the specification.

Example 4, in vitro verification of blood samples taken from healthy humans and diabetic patients with different blood glucose concentrations controls the different brightness of the in-vivo optical module far-red LEDs, thereby inducing different GLP-1 expression levels

This example uses GLP-1 as a reporter gene to demonstrate the different concentrations of blood glucose controllable optical modules. Different brightness thus induces different expression levels of GLP-1, but does not limit the scope of protection of the present invention. Specific steps are as follows:

The fourth step is transfection. After inoculation of the cells for 16 to 24 hours, 4 μg of pWS46, 4 μg of pGY32, 4 μg of pWS212, PEI transfection reagent and serum-free DMEM were mixed, allowed to stand at room temperature for 15 min, and then uniformly added dropwise to a 10 cm cell culture dish. The total volume of each dish was 2 mL, and the mass ratio of plasmid to PEI was 1:3. After 6 h of transfection, 10 mL of DMEM medium containing 10% FBS was exchanged for culture.

In the sixth step, the hydrogel far-red LEDs prepared above were placed in a 24-well plate for culture, and blood samples of different blood glucose concentrations taken from healthy persons and diabetic patients were respectively 6.0 mM, 8.3 mM. , 13.6 mM, 18.4 mM. Dropping on the blood glucose test strip, the blood glucose concentration detector obtains the blood glucose concentration value, and generates blood glucose concentration data; and the blood glucose data processing unit in the blood glucose data automatic control system extracts the blood glucose concentration value from the blood glucose concentration data, according to the blood glucose concentration value Outputting a current corresponding to the voltage to the wireless power supply module; the wireless power supply module outputs a sine wave signal corresponding to the transmit power according to the voltage value of the input current; the optical module includes an inductive receiving coil, a capacitor, a far red LED, and an engineered cell arranged in series The graft carrier; the inductive receiving coil receives the sinusoidal signal to generate an induced current to adjust the luminance of the far-red LED of the optical module, and induces the above-mentioned engineered cells in the hydrogel to generate different GLP-1 expression levels.

The seventh step is to detect the reporter gene. After 48 hours of culture, the cell culture supernatant of each group was used to determine the expression level of GLP-1 (specific methods refer to materials and methods).

For example, a blood sample having a blood glucose concentration of 6.0 mM is dropped onto a blood glucose test strip, the concentration is within a threshold range of <6.1 mM, and the brightness of the corresponding far red LED is 0 mW/cm ² , thus 24 holes are measured. The expression level of GLP-1-Fc in the plate supernatant was about 40 pM; blood samples with a blood glucose concentration of 8.3 mM were added to the blood glucose test strip, and the concentration was within the threshold range of 6.1-11.1 mM, corresponding to the far red The brightness of the light LED is 0.2 mW/cm ² , so that the expression of GLP-1 in the supernatant of the 24-well plate is about 200 pM; the blood sample with the blood glucose concentration of 13.6 mM is added to the blood glucose test strip. In the threshold range of 11.1-16.8 mM, the brightness of the corresponding far-red LED is 1.0 mW/cm ² , so that the GLP-1 in the 24-well plate is about 400 pM; the blood glucose is 18.4 mM. The sample was added dropwise to the blood glucose test strip. The concentration was within the threshold range of >16.8 mM, and the brightness of the corresponding far red LED was 5.0 mW/cm ² . Thus, the GLP-1 in the supernatant of the 24-well plate was measured. It is 570pM.

The results show that the blood glucose data automatic control system in the diabetes ultra-long-range intelligent diagnosis and treatment system can output the corresponding voltage current to the wireless power supply module according to the input blood sample with different blood glucose concentration thresholds (taken from healthy people and diabetic patients), the induction of the optical module The receiving coil receives the sinusoidal signal to generate an induced current to adjust the luminance of the far-red LED of the optical module, and induces the above-mentioned engineered cells in the hydrogel to generate different GLP-1-Fc expression levels. In this example, GLP-1-Fc was used as a reporter gene to verify the function of the diabetes ultra-long-range intelligent diagnosis and treatment system in vitro. The experimental data is detailed in Figure 13 of the specification.

Example 5, using SEAP as a reporter gene, to verify the digital self-reward function of the diabetes ultra-long-range intelligent diagnosis and treatment system for different blood glucose concentrations in wild type mice and type II diabetic mice.

In this example, SEAP is used as a reporter gene. It is exemplified that the different blood glucose concentrations of different mice can control the different brightness of the optical module far-red LEDs implanted in each mouse, thereby inducing different SEAP expression levels, but not for the present invention. The scope of protection is limited. Specific steps are as follows:

In the third step, the cells are inoculated. The well-prepared HEK-293 cells were digested with 0.25% trypsin and seeded in 10 cm cell culture dishes at 4 x 10 ⁶ cells per dish, and 10 mL of DMEM medium containing 10% FBS was added.

In the sixth step, the hydrogel far-red LEDs prepared above were transplanted into wild type mice and type II diabetes. In the abdominal cavity of the mouse, the mice were subjected to blood sampling from the tail vein, and the blood samples obtained were 5.9 mM, 9.4 mM, 14.2 mM, and 17.2 mM, respectively. Dropping on the blood glucose test strip, the blood glucose concentration detector obtains the blood glucose concentration value, and generates blood glucose concentration data; and the blood glucose data processing unit in the blood glucose data automatic control system extracts the blood glucose concentration value from the blood glucose concentration data, according to the blood glucose concentration value Outputting a current corresponding to the voltage to the wireless power supply module; the wireless power supply module outputs a sine wave signal corresponding to the transmit power according to the voltage value of the input current; the optical module includes an inductive receiving coil, a capacitor, a far red LED, and an engineered cell arranged in series The graft carrier; the inductive receiving coil receives the sinusoidal signal to generate an induced current to adjust the luminance of the far-red LED of the optical module, and induces the above-mentioned engineered cells in the hydrogel to generate different SEAP expression levels. The seventh step is to detect the reporter gene. After 48 hours of operation in the self-feedback system of the diabetes ultra-long-range intelligent diagnosis and treatment system, the mice were subjected to intraocular blood sampling, and the expression of SEAP in the blood of the mice was determined (specific methods and materials).

For example, a blood sample having a blood glucose concentration of 5.9 mM is dropped onto a blood glucose test strip, the concentration is within a threshold range of <6.1 mM, and the brightness of the corresponding far red LED is 0 mW/cm ² , thus 24 holes are measured. The amount of SEAP in the supernatant was about 10 mU/L; the blood sample with a blood glucose concentration of 9.4 mM was added to the blood glucose test strip, and the concentration was within the threshold range of 6.1-11.1 mM, corresponding to the far red LED. The brightness was 0.2 mW/cm ² , so the SEAP expression in the clear solution of the 24-well plate was measured to be about 100 mU/L; the blood sample with the blood glucose concentration of 14.2 mM was added to the blood glucose test strip at a concentration of 11.1. Within a threshold range of -16.8 mM, the brightness of the corresponding far-red LED is 1.0 mW/cm ² , so that the SEAP in the 24-well plate is about 200 mU/L; the blood sample with a blood glucose concentration of 17.2 mM is dropped. Add to the blood glucose test strip, the concentration is within the threshold range of >16.8 mM, and the brightness of the corresponding far red LED is 5.0 mW/cm ² , thus measuring the SEAP in the clear solution of the 24-well plate is about 300 mU/L. .

The results show that the blood glucose data automatic control system in the diabetes ultra-long-range intelligent diagnosis and treatment system can output the corresponding voltage current to the wireless power supply module according to the input blood sample with different blood glucose concentration thresholds (taken from wild type mice and type II diabetic mice). The inductive receiving coil of the optical module receives the sinusoidal signal to generate an induced current to adjust the luminance of the far-red LED of the optical module, and induces the above-mentioned engineered cells in the hydrogel to generate different SEAP expression levels. In this example, SEAP was used as a reporter gene to verify the function of the diabetes ultra-long-range intelligent diagnosis and treatment system in type II diabetic mice. The experimental data is detailed in Figure 14 of the specification.

Example 6, verification of diabetes ultra-long-range intelligent diagnosis and treatment system in type II diabetic mice for II Diagnostic and therapeutic functions of type 2 diabetes

In this embodiment, a type II diabetic mouse is taken as an example to demonstrate the diagnosis and treatment function of the diabetes ultra-long-range intelligent diagnosis and treatment system for diabetes, but the scope of protection of the present invention is not limited. Specific steps are as follows:

The sixth step is to determine the in vivo experimental process and experimental results. The hydrogel far-red LED prepared above was transplanted into the abdominal cavity of wild type mice and type II diabetic mice (5, numbered 1, 2, 3, 4, 5, respectively) that had been fasted for 8 hours. After 1 hour of transplantation (this time is recorded as the 0th hour), the mice were subjected to blood sampling in the tail vein and the eyeball, and the blood sample in the eye was used to measure the amount of GLP-1 in the blood of the mouse. The blood sample obtained from the tail vein is added to the blood glucose test strip, and the blood glucose concentration detector obtains the blood glucose concentration value, and the blood glucose concentration data is generated; and the blood glucose concentration is extracted from the blood glucose concentration data by the blood glucose data processing unit in the blood glucose data automatic control system. The value outputs a current corresponding to the voltage according to the blood glucose concentration value to the wireless power supply module; the wireless power supply module outputs a sine wave signal corresponding to the transmission power according to the voltage value of the input current; the optical module includes an inductive receiving coil, a capacitor, and a far red light arranged in series LED and a graft carrier containing engineered cells; the inductive receiving coil receives a sinusoidal signal to generate an induced current to adjust the luminance of the far-red LED of the optical module, and induces the above-mentioned engineering in the hydrogel The cells produced different levels of GLP-1 expression, and recorded the brightness of the far-red LED. After 4 hours of irradiation with the far-red LED, the illumination was stopped until the 16th hour, and then fasted for 8 hours. At the end of fasting, it was the 24th hour. The mice were then subjected to blood sampling in the tail vein and the eyeball, and blood samples were taken from the eye to measure the amount of GLP-1 in the blood of the mice. The blood sample taken from the tail vein was added to the blood glucose test strip. Data transmission through the operation of the diabetes ultra-long-range intelligent diagnosis and treatment system To the optical module output corresponding brightness of the far red LED (the same principle is the same as above, no longer repeated here), and record the brightness of the far red LED, after 4 hours of illumination by the far red LED, stop the illumination until the 40th hour, then ban 8h, the end of fasting is the 48th hour. At this time, the mice are again subjected to blood sampling in the tail vein and the eyeball. The blood sample in the eye is used to measure the amount of GLP-1 in the blood of the mouse. The blood sample obtained from the tail vein is added to the blood glucose test strip, and the data is transmitted to the optical module to output the corresponding far red LED brightness through the operation of the diabetes ultra-long-range intelligent diagnosis and treatment system (the specific principle is the same as above, and is not repeated here). And record the brightness of the far red LED, after 4 hours of irradiation with the far red LED, stop the light until the 53th hour, then fast for 8h, the end of the fast is 72 hours, then measure the blood sugar value of the 72nd hour and The amount of GLP-1 in the blood. The expression level of GLP-1 in the blood of the mouse was measured (specific methods refer to materials and methods).

In this experiment, three sets of data were recorded simultaneously, namely the time, the blood glucose concentration of the mouse at each time period, and the brightness of different far-red LEDs excited by the diabetes ultra-long-range intelligent diagnosis and treatment system. For example, at 0h, the blood glucose concentrations of

mice

1, 2, 3, and 4 are all above 18 mM. Through the diabetes ultra-long-range intelligent diagnosis and treatment system, the brightness of the far-red LEDs excited is 5.0 mW/cm ² , 5 The blood glucose concentration is 15 mM. Through the diabetes ultra-long-range intelligent diagnosis and treatment system, the brightness of the far-red LED that is excited is 1.0 mW/cm ² , which in turn produces different degrees of hypoglycemic effect. The blood glucose concentration was detected at 24h, and the blood glucose concentration of No.1 and No.4 had dropped to about 14 mM. Through the ultra-long-range intelligent diagnosis and treatment system for diabetes, the brightness of the far-red LED was 1.0mW/cm ² , and the blood glucose concentration of 3 was reduced to 10.5. About mM, through the diabetes ultra-long-range intelligent diagnosis and treatment system, the brightness of the far-red LED that is excited is 0.2mW/cm ² , and the blood glucose concentration of ² , 5 has dropped to about 18mM, which is stimulated by the diabetes ultra-long-range intelligent diagnosis and treatment system. The brightness of the far-red LED was 5.0 mW/cm ² . The blood glucose concentration was detected at 48h, and the blood glucose concentration of 1, 3, and 5 was reduced to 8.5 mM. The brightness of the far-red LED excited by the ultra-long-range intelligent diagnosis and treatment system of diabetes was 0.2mW/cm ² , and the blood glucose concentration of ² and 4 was obtained. The brightness of the far-red LED excited by the diabetes ultra-long-range intelligent diagnosis and treatment system was reduced to about 14 mM, which was 1 mW/cm ² . The blood glucose concentration was detected at 72h, and the blood glucose concentration of 1, 3, 4, and 5 was reduced to about 8.5 mM. The brightness of the far-red LED excited by the ultra-long-range intelligent diagnosis and treatment system of diabetes was 0.2mW/cm ² , and the blood glucose concentration of No. 2 was obtained. The brightness of the far-red LED excited by the diabetes ultra-long-range intelligent diagnosis and treatment system was reduced to about 13 mM, which was 1 mW/cm ² . The average expression of GLP-1 in blood of 24h, 48h and 72h mice was 45pM, 65pM and 65pM, respectively. In the control group, the far-red LED in the normal blood glucose mice was always in the closed state, and the expression level of GLP-1 in the blood was always at a low level, about 12 pM.

The results show that the blood glucose data automatic control system in the diabetes ultra-long-range intelligent diagnosis and treatment system can output the current of the corresponding voltage to the wireless power supply module according to the blood sugar level of the type II diabetic mouse, and the induction receiving coil of the optical module receives the sine wave signal and generates the induction. The current is adjusted to reduce the luminescence brightness of the optical module far red LED, and the above-mentioned engineered cells in the hydrogel are induced to produce different GLP-1 expression levels to reduce the blood glucose concentration in the type II diabetic mice, which is compared with the control group. Significant differences, hyperglycemia symptoms were significantly alleviated, blood glucose levels were basically reduced to normal levels. In this embodiment, the hypoglycemic drug GLP-1 is used as a reporter gene, and the optical module of the diabetes ultra-long-range intelligent diagnosis and treatment system is transplanted into the type II diabetic mouse, and the diagnosis of the diabetes ultra-long-range intelligent diagnosis and treatment system is verified in the type II diabetic mouse. And the therapeutic function, that is, after measuring blood sugar, the system can accurately regulate the expression level of glucagon-like peptide (GLP-1) according to its blood sugar level, and carry out targeted treatment, avoiding the hypoglycemic excess caused by the traditional hypoglycemic method. The adverse consequences brought about, thus achieving the purpose of automation, precision and individualized treatment of diabetes. Through the self-diagnosis and treatment functions of this system, the blood glucose of type II diabetic mice basically returned to normal levels. The experimental data is detailed in Figures 15, 16, 17 of the specification.

Table 1 plasmid construction table

Second, far red light regulation gene expression loop control system

Materials and Methods:

All primers used for PCR were synthesized by Jinweizhi Biotechnology Co., Ltd. The expression plasmids in the examples of the present invention are all carried out according to a conventional molecular cloning procedure, and the constructed expression plasmids are all sequenced, and the sequence determination is performed by Jinweizhi Biotechnology Co., Ltd. The DNA polymerase, endonuclease and T4 DNA ligase used in the examples of the present invention were all purchased from Nanjing Nuoweizan Biotechnology Co., Ltd. The dual luciferase reporter assay kit used was purchased from Biotool, USA. Hollow fiber membrane graft tube used in the experiment

Implant Membrane) purchased Spectrum Laboratories, Inc., USA. The Mouse Insulin ELISA kit used in the experiment was purchased from Mercodia, Sweden. The glucagon assay kit (Millipore Corporation, Billerica, MA 01821 USA, Cat. no. EGLP-35K, Lot. no. 2639195) used in the experiment was purchased from Millipore Corporation, USA.

Example 7, Construction of a Gene Loop Regulatory System Element for Far Red Light Regulating Transgene Expression

In this embodiment, a method for constructing representative elements in a gene loop remote control system for far-red light regulation of transgene expression is included, but the scope of protection of the present invention is not limited. The detailed design scheme and steps are shown in Table 2.

Example 8, far red light regulates transgene expression of far red light regulation gene expression loop control system photoreceptor expressed by different promoters

The first step is plasmid construction. The plasmid construction in this example is shown in Table 2. In the second step, the cells are inoculated. The third step is transfection. Two 24-well plates were divided into dark and light groups within 16 to 24 hours of seeding, and each group was divided into 1-5 groups. Within 1 to 24 hours of seeding the cells, 0.1 μg of pWS50 (expressed by the promoter SV40) was added to the 1st group of the dark and light groups, and 0.1 μg of pWS189 (expressed by the promoter hCMV) in the second group, Group 3 0.1 μg of pWS51 (expressed by the promoter hEF1α), 0.1 μg of pWS55 (expressed by the promoter mPGK) in the fourth group, and 0.1 μg of pWS59 (expressed by the promoter CAG) in the fifth group and 0.01 μg, respectively The pSTING, 0.1 μg effector pWS67, PEI transfection reagent were mixed with serum-free DMEM, allowed to stand at room temperature for 15 min, and then uniformly added dropwise to a 24-well culture plate. The fourth step, illumination, after changing liquid for 14-18h, the illumination group was placed on a LED with a wavelength of 720 nm and an illumination intensity of 1 mW/cm ² for 4 h. The fifth step is to detect the reporter gene.

As shown in Fig. 20, in the gene loop control system of far red light regulating transgene expression, photoreceptors expressed by different promoters can work normally in mammalian cells under the induction of far red light. However, the photoreceptors expressed by different promoters under the same far red light induce different reaction intensity of the effector. From the experimental results, the photoreceptor induced by the promoter CMV has the highest fold induction.

Example 9, a far-red light-regulated gene loop control system for different types of processors

The first step is plasmid construction. The plasmid construction in this example is detailed in Table 2. In the second step, the cells are inoculated. The third step is transfection. Two 24-well plates were divided into dark and light groups within 16 to 24 hours of seeding, and each group was divided into 1-8 groups. Within 16 to 24 hours of inoculation of cells, 0ng pSTING in the first group of the dark group and the light group, 5ng pSTING in the second group, 10ng pSTING in the third group, and 20ng pSTING in the fourth group, the fifth group Lieutenant 40 ng pSTING, group 6 60 ng pSTING, group 7 80 ng pSTING, group 8 100 ng pSTING and 100 ng far red photoreceptor pWS189, 100 μg effector pWS67, PEI transfection reagent and serum free The mixture was mixed in DMEM, allowed to stand at room temperature for 15 min, and then uniformly added dropwise to a 24-well culture plate. The fourth step, illumination (the specific steps are the same as in Example 2). The fifth step is to detect the reporter gene.

As a result, as shown in Fig. 21, in the gene loop control system in which the far red light regulates the expression of the transgene, different amounts of the processor cause the effect intensity of the effector to be different under the same far red light induction. When the processor is 10ng, the photoreceptor is 100ng, the effector is 100ng, the expression ratio is the highest, and the background is low (SEAP expression is only 5-8U/L), the processor is 40ng, the photoreceptor is 100ng, the effector The expression level is the highest at 100 ng but the expression ratio is not high at 10 ng due to the higher background; the optimal processor amount can be selected according to experimental needs.

Example 9, a processor constructed by a different gene loop control system for far-red light regulation of transgene expression

In the present embodiment, a processor constructed by a different example of a gene loop control system for demonstrating far-red light regulation of transgene expression is regulated by far-red light in mammalian cells, but does not limit the scope of protection of the present invention. Specific steps are as follows:

The first step is plasmid construction. The plasmid construction in this example is detailed in Table 2. In the second step, the cells are inoculated. The third step is transfection. Two 24-well plates were divided into dark and light groups within 16 to 24 hours of seeding, and each group was divided into 1-8 groups. Within 1 to 24 hours of inoculation of cells, 0.1 μg of pWS200 in the first group of the dark group and the light group, 0.1 μg of pXY24 in the second group, 0.1 μg of pXY35 in the third group, and 0.1 μg of pXY36 in the fourth group In the fifth group, 0.1 μg of pGY28, the sixth group of 0.1 μg of pGY32, the seventh group of 0.1 μg of pGY33, the eighth group of 0.1 μg of pGY34 and 0.01 μg of far-red photoreceptor pWS189, and 0.1 μg of effect. The pXY24 and PEI transfection reagents were mixed with serum-free DMEM, allowed to stand at room temperature for 15 min, and then uniformly added dropwise to a 24-well culture plate. The total volume of preparation per well was 50 μL, and the mass ratio of plasmid to PEI was 1:3. After 6 h of transfection, 500 μL of DMEM medium containing 10% FBS was exchanged for culture. The fourth step, illumination (the specific steps are the same as in Example 7). In the fifth step, the reporter gene is detected (the specific steps are the same as in Example 7).

The results show that in the gene loop control system of far red light regulating transgene expression, different processors can work normally in mammalian cells under far-red light induction. However, differently constructed processors under the same far-red light induction make the effector's reaction intensity different, and different processors can be selected according to the experimental needs. The experimental data is detailed in Figure 22 of the specification.

Example 10, differently constructed effectors of the gene loop control system for far-red light regulation of transgene expression

The first step is plasmid construction. The plasmid construction in this example is detailed in Table 2. In the second step, the cells are inoculated. The third step is transfection. Two 24-well plates were divided into dark and light groups within 16 to 24 hours of seeding, and each group was divided into 1-9 groups. Within 1 to 24 hours of seeding the cells, 0.1 μg of pWS32 was placed in the first group of the dark group and the light group, 0.1 μg of pWS33 in the second group, 0.1 μg of pWS35 in the third group, and 0.1 μg of pWS54 in the fourth group. In the fifth group, 0.1 μg of pWS58, the sixth group of 0.1 μg of pWS67, the seventh group of 0.1 μg of pYW25, the eighth group of 0.1 μg of pYW28, and the ninth group of 0.1 μg of pYW29, and 0.1 μg, respectively. The far-red photoreceptor pWS189, 0.01 μg processor pSTING, PEI transfection reagent and serum-free DMEM were mixed, allowed to stand at room temperature for 15 min, and then uniformly added dropwise to a 24-well culture plate. The fourth step, illumination (the specific steps are the same as in Example 7). The fifth step, check Report the gene.

The results are shown in Figure 23, in the gene loop control system for far-red light regulation of transgene expression, pWS32 (P _{FRL1 (5 × ISRE-h_CMVmin)} ), pWS33 (P _{FRL2 (hIFN-RE-h_CMVmin)} ), pWS35 (P _{FRL3 ((hIFN-RE)-3} _{× ISRE-h_CMVmin)} ), pWS54 (P _{FRL4 ((hIFN-RE)-3×ISRE-h_min)} ), pWS58 (P _{FRL5 ((hIFN-RE)-3×} _{ISRE- (hIFN-RE)-3×ISRE-h_min)} ), pWS67 (P _{FRL6 ((hIFN-RE)-3×ISRE-h_min-40bp)} ), pYW25 (P _{FRL7((hIFN-RE)-h_min)} ), pYW28 (P _{FRL8 ((hIFN-RE)-3×ISRE-(hIFN-RE)-h_min)} ), pYW29 (P _{FRL9 (3} _{× ISRE-(hIFN-RE)-h_min)} ), different processor identification bits The different repeats of the spots and recognition sites and the effectors of different kinds of weak promoters can work normally in mammalian cells under far-red light induction. However, under the same far-red light induction, the response intensity of different effectors including different processor recognition sites and different recognition sites is different. According to the experimental results, pWS67 is found as the effector with the highest expression ratio, while pWS35 As the effector, the expression level is the highest, and different effectors can be selected according to the experimental needs. Example 11, a different constructed effector of a gene loop control system for far-red light regulation of transgene expression

In this example, a different constructed effector of a gene loop control system for demonstrating far-red light regulation of transgene expression is regulated by far red light in mammalian cells. Taking different processor recognition sites, different repetition numbers of recognition sites, whether there is insulation signal and different kinds of weak promoters as examples, different effectors are regulated by far red light in mammalian cells, but The scope of protection of the present invention is not limited.

Different processor recognition sites and different repetition numbers of recognition sites. The specific steps are as follows:

The first step is plasmid construction. The plasmid construction in this example is detailed in Table 2. In the second step, the cells are inoculated. The third step is transfection. Two 24-well plates were divided into dark and light groups within 16 to 24 hours of seeding, and each group was divided into 1-10 groups. Within the first 16 to 24 hours of inoculation of cells, 0.1 μg of pXY19 in the first group of the dark group and the light group, 0.1 μg of pXY20 in the second group, 0.1 μg of pXY21 in the third group, and 0.1 μg of pXY22 in the fourth group 0.1 μg pXY23 in the fifth group, 0.1 μg pXY16 in the sixth group, 0.1 μg pXY17 in the seventh group, 0.1 μg pXY18 in the eighth group, and 0.1 μg pXY31 in the ninth group, in the 10th group. 0.1 μg of pXY32 and 0.1 μg of far-red photoreceptor pWS189, 0.1 μg of processor pGY32, PEI transfection reagent and serum-free DMEM were mixed, allowed to stand at room temperature for 15 min, and then uniformly added dropwise to a 24-well culture plate. The total volume of preparation per well was 50 μL, and the mass ratio of plasmid to PEI was 1:3. After 6 h of transfection, 500 μL of DMEM medium containing 10% FBS was exchanged for culture. The fourth step, illumination (the specific steps are the same as in Example 7). The fifth step, detecting the reporter gene (specific steps The same as Example 7).

The results showed that in the gene loop control system of far red light regulating transgene expression, different processor recognition sites and effectors of different repeat number recognition sites can work normally in mammalian cells under far red light induction. . However, the response intensity of different processor recognition sites and different repeat number recognition sites under different far-red light induction is different, and different processors can be selected according to experimental needs. The experimental data is detailed in Figures 24 and 25 of the specification.

Add an insulation signal before the processor identification site with different repetition numbers. Specific steps are as follows:

The first step is plasmid construction. The plasmid construction in this example is detailed in Table 2. In the second step, cells were seeded (the specific steps are the same as in Example 7). The third step is transfection. Two 24-well plates were divided into dark and light groups within 16 to 24 hours of seeding, and each group was divided into 1-5 groups. Within 1 to 24 hours of seeding the cells, 0.1 μg of pXY33 was placed in the first group of the dark group and the light group, 0.1 μg of pXY28 was used in the second group, 0.1 μg of pXY34 was used in the third group, and 0.1 μg of pXY39 was used in the fourth group. In the fifth group, 0.1 μg of pXY40 and 0.1 μg of far-red photoreceptor pWS189, 0.1 μg of processor pGY32, PEI transfection reagent and serum-free DMEM were mixed, allowed to stand at room temperature for 15 min, and then uniformly added to 24-well culture. In the board. The total volume of preparation per well was 50 μL, and the mass ratio of plasmid to PEI was 1:3. After 6 h of transfection, 500 μL of DMEM medium containing 10% FBS was exchanged for culture. The fourth step, illumination (the specific steps are the same as in Example 7). In the fifth step, the reporter gene is detected (the specific steps are the same as in Example 7).

The results show that in the gene loop control system of far red light regulating transgene expression, the effector with the insulation signal added before the processor recognition site containing different repeat numbers can be normal in mammalian cells under far red light induction. jobs. However, the effect intensity of an effector that adds an insulation signal before the processor identification site with different repetition numbers under the same far-red light induction is different, and different processors can be selected according to experimental needs. The experimental data is detailed in Figure 26 of the specification.

Different kinds of weak promoters. Taking the promoter h_CMV _min3G as an example, it is possible to use different weak promoters. Specific steps are as follows:

The first step is plasmid construction. The plasmid construction in this example is detailed in Table 2. In the second step, cells were seeded (the specific steps are the same as in Example 7). The third step is transfection. Two 24-well plates were divided into dark and light groups within 16 to 24 hours of seeding, and each group was divided into 1-5 groups. Within 1 to 24 hours of seeding the cells, 0.1 μg of pGY36 was placed in the first group of the dark group and the light group, 0.1 g of pGY37 was used in the second group, 0.1 μg of pGY38 was used in the third group, and 0.1 μg of pGY39 was used in the fourth group. In the fifth group, 0.1μg pGY40 0.1 μg of far-red photoreceptor pWS189, 0.1 μg of processor pGY32, PEI transfection reagent and serum-free DMEM were separately mixed, allowed to stand at room temperature for 15 min, and then uniformly added dropwise to a 24-well culture plate. The total volume of preparation per well was 50 μL, and the mass ratio of plasmid to PEI was 1:3. After 6 h of transfection, 500 μL of DMEM medium containing 10% FBS was exchanged for culture. The fourth step, illumination (the specific steps are the same as in Example 7). In the fifth step, the reporter gene is detected (the specific steps are the same as in Example 7).

The results show that in the gene loop control system of far red light regulating transgene expression, different weak promoters contained in the effector can work normally in mammalian cells under far red light induction. However, the weak promoter is not as strong as other weak promoters. The experimental data is detailed in Figures 25 and 27 of the specification.

Example 12, the gene loop control system of far red light regulating transgene is expressed in different mammalian cells

The first step is plasmid construction. The plasmid construction in this example is detailed in Table 2. In the second step, the cells are inoculated. The third step is transfection. Within 16 to 24 hours after seeding the cells, 0.1 μg of pWS189, 0.01 μg of pSTING, 0.1 μg of pWS67, PEI transfection reagent and serum-free DMEM were mixed, allowed to stand at room temperature for 15 min, and then uniformly added dropwise to a 24-well culture plate. The fourth step, illumination (the specific steps are the same as in Example 7). The fifth step is to detect the reporter gene.

As a result, as shown in Fig. 28, the gene loop control system for far-red light-regulated transgene expression in the present invention can be subjected to different mammalian cells (e.g., hMSC-TERT, Hana 3A, HEK-293A, HEK-293T). Far red light induces expression. Therefore, the gene loop control system of the far-red light-regulated transgene in the present invention can be expressed in various mammalian cell types, and can be applied to various mammalian cells.

Example 13, controlling different illumination times to regulate different expression levels of gene loop control systems for far red light regulating transgene expression

The first step is plasmid construction. The plasmid construction in this example is detailed in Table 2. In the second step, the cells are inoculated. The third step is transfection. Within 16 to 24 hours after seeding the cells, 0.1 μg of photoreceptor pWS189, 0.01 μg of pSTING, 0.1 μg of pWS67, PEI transfection reagent and serum-free DMEM were mixed, allowed to stand at room temperature for 15 min, and then uniformly added dropwise to a 24-well culture plate. The fourth step is to control different lighting times for illumination. After changing the liquid for 14-18 hours, it was divided into 13 groups and placed under an LED having a wavelength of 720 nm and an illumination intensity of 1 mW/cm ² . The different illumination times were 0, 0.01, 0.1, 0.25, 0.5, 1, 2, 4, 6, 12, 24, 48, 72 h (where the group with 0 h of illumination was kept in the dark). The fifth step is to detect the reporter gene. After 72 h of culture, the cell culture supernatants of each group were taken to determine the expression level of SEAP.

The experimental results are shown in Fig. 29. By controlling different illumination time, the gene loop control system of the far red light regulating transgene can be induced to regulate the different expression levels of the target gene, and the longer the light induction time, the higher the expression amount is at 0- Illumination time-dependent expression was presented within 72 h.

Example 14, Controlling Different Expression Levels of Gene Loop Control Systems Regulating Transgene Expression by Far Red Light by Controlling Different Light Intensities

The first step is plasmid construction. The plasmid construction in this example is detailed in Table 2. In the second step, the cells are inoculated. The third step, transfection (specific steps are the same as in Example 13). The fourth step is to control different light intensities. After changing liquid for 14-18h, it was divided into 11 groups and placed at LEDs with wavelength of 720nm and light intensity of 0, 25, 50, 75, 100, 250, 500, 750, 1000, 1500, 2000μW/cm ² respectively. under. The illumination time was 4 h (the group with the light intensity of 0 was kept in the dark). The fifth step is to detect the reporter gene. After 72 hours of culture, the cell culture supernatants of each group were taken to determine the expression level of SEAP.

The experimental results are shown in Figure 30. The control of different light intensities can induce the different expression levels of the target genes in the gene loop control system that regulates the expression of transgenes by far red light, and the stronger the light intensity, the higher the expression level, within 0-72h. Light intensity dependent expression is presented.

Example 15, a gene loop control system that regulates transgenes can express all meaningful proteins

The first step is plasmid construction. The plasmid construction in this example is detailed in Table 2. In the second step, the cells are inoculated. The third step is transfection. Within 16 to 24 hours of inoculation of cells, 0.1 μg of pWS189, 0.01 μg of pSTING, 0.1 μg of pGY45 (expressing Luciferase); 0.1 μg of pWS189, 0.01 μg of pSTING, 0.1 μg of pWS152 (expressing GLP-1-FC) and PEI transfection reagent, respectively The serum-free DMEM was mixed, allowed to stand at room temperature for 15 min, and then uniformly added dropwise to a 24-well culture plate. The fourth step, illumination (specific steps are the same as in Example 7), the expression of Luciferase was measured after 4 hours of illumination; GLP-1 was uniformly determined immediately after 0, 0.1, 0.25, 0.5, 1, 2 hours of illumination. The amount of expression at different illumination times. The dark group was kept in the dark for cultivation. The fifth step is to detect the reporter gene. The expression level of luciferase (Luciferase) was determined by ELISA kit at 24h and 48h (Fig. 31). The expression of GLP-1 at different illumination time was determined by ELISA kit at 48h (Fig. 32). ).

As a result, as shown in Figs. 31 and 32, the gene loop control system for regulating transgene expression of the present invention can well induce expression of different proteins such as Luciferase, GLP-1-Fc, etc., and there is no particular limitation on the kind of protein. Therefore, the gene loop control system for regulating the transgene of the present invention is suitable for expressing all meaningful meanings. Protein.

At the same time, as can be seen from Figures 33 and 34, the gene loop control system regulating the expression of transgene can precisely regulate the expression of EGFP and insulin, and its expression level is positively correlated with the illumination time.

Example 16, a gene loop control system that regulates transgenes can simultaneously express two or more proteins of all significance

The first step is plasmid construction. The plasmid construction in this example is detailed in Table 2. In the second step, the cells are inoculated. The third step is transfection. Within 16 to 24 hours after seeding the cells, 0.1 μg of pWS189, 0.01 μg of pSTING, 0.1 μg of pWS174, PEI transfection reagent and serum-free DMEM were mixed, allowed to stand at room temperature for 15 min, and then uniformly added dropwise to a 24-well culture plate. The fourth step is lighting. After changing the liquid for 14-18 h, the illumination group was placed at a wavelength of 720 nm, and the set illumination intensity of 1 mW/cm ² was uniformly measured immediately after 0, 0.1, 0.25, 0.5, 1, 2 hours. The fifth step is to detect the reporter gene.

Results As shown in Figures 33 and 34, the gene loop control system for regulating transgene expression of the present invention can express two different proteins (connected with 2A) at the same time, and the two different proteins simultaneously expressed have Time dependence. Thus, the gene loop control system of the regulatory transgene of the present invention can be used to simultaneously express two or more significant proteins.

At the same time, as shown in Fig. 34, the gene loop control system regulating the expression of transgene can precisely regulate insulin expression in vitro, and its expression level is positively correlated with the illumination time.

Example 17: Preparation of a hollow fiber membrane graft tube grafting vector containing engineered cells containing a gene loop control system that regulates expression of a transgene

The first step is plasmid construction. The plasmid construction in this example is detailed in Table 2. The second step, inoculate the cells. The third step, transfection: within the period of 16 to 24 hours after inoculation of cells, 0.1μg pWS189, 0.01μg pSTING, 0.1μg pWS67, PEI transfection reagent and serum-free DMEM are mixed, and allowed to stand at room temperature. After 15 min, it was evenly added dropwise to a 24-well cell culture plate. After 6 h of transfection, 10 mL of DMEM medium containing 10% FBS was exchanged for culture. The fourth step is to prepare a hollow fiber membrane graft tube for engineering cells of a gene loop control system that regulates transgene expression. After changing for 14-18 h, the cells were trypsinized and centrifuged to collect the cells. A hollow fiber membrane graft tube was produced according to the production method.

The experimental results are shown in Fig. 35. The prepared hollow fiber membrane graft tube, the nutrients required for cell growth, and the small molecule protein of interest secreted by the engineered cells can pass through the membrane system freely. But cells and other macromolecular proteins cannot pass through the membrane system. Therefore, the cells wrapped by the hollow fiber membrane graft can Transplanted into mice to grow normally.

Example 18, toxicity test of far red light on cells

The first step is plasmid construction. The plasmid construction in this example is detailed in Table 2. In the second step, the cells are inoculated. The third step is transfection. Within 16 to 24 hours of seeding the cells, 0.1 μg of pSEAP2 control and PEI transfection reagent were mixed with serum-free DMEM, allowed to stand at room temperature for 15 min, and then uniformly added dropwise to a 24-well culture plate. After 6 h of transfection, 500 μL of DMEM medium containing 10% FBS was exchanged for culture. The fourth step is lighting. After changing the liquid for 14-18h, it is divided into 10 groups, which are respectively placed at the wavelength of 720nm, and the illumination with the set light intensity of 1mW/cm ² is 0, 0.1, 0.5, 1, 2, 6, 12, 24, 48, 72 hours immediately after the unified measurement. The fifth step is to detect the reporter gene.

The experimental results are shown in Fig. 36. After 72 hours of illumination, the expression level of SEAP is basically the same as that of darkness, indicating that far red light is not toxic to cells.

Example 19, background measurement of the far red light regulating gene expression loop control system of the present invention

The first step is plasmid construction. The plasmid construction in this example is detailed in Table 2. In the second step, the cells are inoculated. The third step is transfection. Within 16 to 24 hours of seeding the cells, 0.1 μg of pWS189, 0.01 μg of pSTING, 0.1 μg of pWS67, 0.1 μg of pWS189, 0.1 μg of pGY32, and 0.1 μg of pXY34 were mixed with PEI transfection reagent and serum-free DMEM, respectively, and allowed to stand at room temperature for 15 min. After that, it was evenly added dropwise to a 24-well culture plate. In the fourth step, after transfection for 6 hours, 500 μL of DMEM medium containing 10% FBS was exchanged, and wrapped in tin foil paper and placed in an incubator for culture. The fifth step is to detect the reporter gene. After 48 hours of culture, the cell culture supernatant of each group was taken to determine the expression level of SEAP.

The experimental results are shown in Fig. 37. After 72 hours of culture, the expression levels of SEAP in the two groups can be seen that the background of the far red light regulation gene expression loop control system of the present invention is low, and the expression level of SEAP is only 5-8 U/L.

Example 20, a gene loop control system regulating transgene expression is regulated by far red light in mice

In the first step, a hollow fiber membrane graft tube was prepared (refer to Example 17 in a specific manner). In the second step, a hollow fiber membrane graft tube was implanted into the back of the mouse. The third step is lighting. The infrared therapeutic device (10 mW/cm ² ) was irradiated for 2 hours at 2h, 8h, 26h and 32h after transplantation. The fourth step is to detect the reporter gene. The amount of reporter gene was determined by blood sampling at 24 h and 48 h after transplantation, respectively.

The experimental results are shown in Fig. 38, indicating that the gene loop control system of the present invention is also controlled by far red light in mice, and is time dependent.

Example 21: Gene loop control system regulating transgene expression accurately regulates insulin expression in type I diabetes model mice for type I diabetes

The first step is the construction of a mouse model of type I diabetes. We used multiple low-dose streptozotocin (Streptozocin, STZ, purchased from Sigma S0130, 18883-6, 6-4) to induce the model. 25 C57BL/6J mice (from Chinese Academy of Sciences), 8 weeks old, male, intraperitoneal for 5 consecutive days (fasting 12-16h before injection) were injected with sodium citrate buffer dissolved in STZ (dose 40-50mg/kg) liquid. In the second step, the preparation process of the engineered cells of the gene loop control system regulating the expression of the transgene is specifically referred to in Example 10. In the third step, the preparation of the hollow fiber membrane graft tube of the engineered cells of the gene loop control system for regulating the expression of the transgene is specifically referred to in Example 17. In the fourth step, the gene loop control system that regulates the expression of the transgene is engineered into the transplanted tube of the mouse implanted into the back of the mouse. The fifth step, illumination, refers specifically to the third step of Embodiment 13. In the sixth step, the fasting blood glucose level of type I diabetic mice was determined. After 8 hours of transplantation, the mice were fasted (water supply) for 16 hours, and blood was taken through the tail to measure the fasting blood glucose level. Experimental data indicate that the expressed insulin has a good hypoglycemic effect (Figure 39). The seventh step, the sugar tolerance test. After 24 hours of transplantation, a glucose tolerance test was performed. Experiments have shown that diabetic mice have a good improvement in glucose tolerance (Figure 40).

Example 22: Gene loop control system regulating transgene expression accurately regulates GLP-1 expression in type 2 diabetes model mice for type II diabetes

The first step is plasmid construction. The plasmid construction in this example is detailed in Table 2. In the second step, 20 type 2 diabetic mouse db/db mice (from Chinese Academy of Sciences), 8 weeks old, female, were divided into four groups. They were not transplanted without light, no light, no light, and light. The third step, the preparation of far red light engineered cells, specifically refer to Example 17. In the fourth step, the preparation of the graft tube of the engineered cells of the gene loop control system for regulating the expression of the transgene is specifically referred to in Example 17. In the fifth step, the gene loop control system for regulating transgene expression controls the transplantation tube of the cells to the back of the mouse, and specifically refer to the second step of the embodiment 21. The sixth step, illumination, refers specifically to the third step of Embodiment 21. In the seventh step, the fasting blood glucose level of type II diabetic mice was determined. After 8 hours of transplantation, the mice were fasted (water supply) for 16 hours, and blood was taken through the tail to measure the fasting blood glucose level. The experimental results are shown in Figure 41. The data indicate that the expressed glucagon has a good hypoglycemic effect. In the eighth step, a glucose tolerance test was performed. After 24 hours of transplantation, a glucose tolerance test was performed. Refer specifically to Example 21 for details. The experimental results are shown in Figure 42, and the glucose tolerance of diabetic rats is very good. In the ninth step, an insulin resistance test was performed. After 24 hours of transplantation, an insulin resistance test was performed. The experimental results are shown in Figure 43, and insulin resistance in diabetic rats is well improved. In the tenth step, the basis for regulating the expression of the transgene is determined. Expression of GLP-1 in type 2 diabetic mice due to loop control system. Blood was taken from the eyeball 48 hours after transplantation, and GLP-1 (activie) content in serum was detected by GLP-1 (7-36) activie ELISA kit. The experimental results are shown in Figure 44. The gene loop control system that regulates the expression of the transgene can precisely regulate the expression of glucagon in vivo.

Table 2. Plasmid construction table

Primers: Enzymatic cleavage sites and seamlessly cloned fragments are underlined.

The protection of the present invention is not limited to the above embodiment. Variations and advantages that may be conceived by those skilled in the art are intended to be included within the scope of the invention and the scope of the appended claims.

Claims

A diabetes ultra-long-range intelligent diagnosis and treatment system, characterized in that the system comprises: an automatic blood sugar data control system, a blood glucose data remote control system, a power supply module, and an optical module;

The blood sugar data automatic control system includes a blood sugar concentration detecting system and a blood sugar data processing unit; the blood sugar data automatic control system generates blood glucose concentration data by acquiring the blood sugar concentration value by the blood sugar concentration detecting system; the blood sugar data processing unit Extracting a blood glucose concentration value from the blood glucose concentration data, and outputting a corresponding voltage and current to the wireless power supply module according to the blood glucose concentration value;

The blood glucose data remote control system includes a mobile device and an intelligent remote controller installed with an application terminal; the mobile device sends an instruction to the intelligent remote controller, and the intelligent remote controller communicates with the blood glucose data processing unit, The blood glucose data processing unit outputs a current corresponding to the voltage according to the instruction;

The wireless power supply module outputs a sine wave signal of a corresponding transmission power according to a voltage value of the input current; the optical module receives an sine wave signal and generates an induced current to adjust a brightness of the LED, thereby inducing the Light-responsive engineered cells express different levels of hypoglycemic agents.
The diabetes ultra-long-range intelligent diagnosis and treatment system according to claim 1, wherein the blood glucose concentration detection system comprises: a blood glucose concentration sensor module, a Bluetooth wireless transmission module, and client software thereof.
The diabetes ultra-long-range intelligent medical treatment system according to claim 2, wherein said blood glucose concentration sensor module is a communication device that can convert blood glucose levels into electrical signals or digital signals.
The diabetes ultra-long-range intelligent diagnosis and treatment system according to claim 1, wherein the blood glucose data processing unit can determine a blood glucose concentration range, and control different relay switches in the relay unit according to the set blood glucose concentration threshold division. The output voltage of the switching power supply of the system is regulated by a relay to output a corresponding current to the wireless power supply module.
The diabetes ultra-long-range intelligent medical treatment system according to claim 1, wherein said blood glucose concentration detecting system transmits blood glucose concentration data to the blood sugar data processing unit via Bluetooth wireless transmission.
The diabetes ultra-long-range intelligent diagnosis and treatment system according to claim 1, wherein said mobile device in said blood glucose data remote control system has a wireless receiving module, said mobile device acquiring blood glucose concentration data and reading by using an application terminal Blood glucose concentration value.
The diabetes ultra-long-range intelligent diagnosis and treatment system according to claim 1, wherein the intelligent remote controller and the mobile device transmit remote control commands through a local area network WiFi or a 2G/3G/4G network to regulate the light source. The device is turned on or off, the light intensity can be adjusted as needed, the lighting time, or the method of illumination.
The diabetes ultra-long-range intelligent diagnosis and treatment system according to claim 1, wherein said power supply mode The block can be any power source that illuminates the LEDs in the optical module, including a low dropout linear regulator chip, an electromagnetic oscillating circuit, a power amplifying circuit, and a transmitting circuit.
The diabetic ultra-long-range intelligent medical treatment system according to claim 1, wherein the optical module comprises an inductive receiving coil, a capacitor, an LED, and a graft carrier containing engineered cells.
The optical module according to claim 9, wherein the LED emitting light comprises violet light, blue light, green light, red light, near-infrared light, and far-red light; and the light-responsive engineered cells are light-induced. Customized cells for gene expression, including far-red, red, green, blue, and ultraviolet light, induce a variety of prokaryotic and eukaryotic cells that regulate gene expression.
The use of the diabetes ultra-long-range intelligent diagnosis and treatment system according to claim 1 in the treatment of diabetes.
A far red light gene loop expression control system, characterized in that the system comprises: a photoreceptor that senses a far red light source; a processor that processes the signal transmitted by the photoreceptor; and a response to the processor The effector of the signal.
The far red light gene loop expression control system according to claim 12, wherein said photoreceptor comprises bacterial photo-sensitive diguanylate cyclase BphS and c-di-GMP degrading enzyme YhjH, said photosensitive The bis-guanylate cyclase BphS is prepared by fusing the amino acids 1-511 of the BphG protein and amino acids 175-343 of the Slr1143 protein, and mutating the 587 arginine of the fusion protein to the alanine R587A. It converts GTP to c-di-GMP under far red light conditions.
The far red light gene loop expression control system according to claim 13, wherein the photoreceptor construction form comprises:

a) synthetic bacteria photosensitive diguanylate cyclase BphS encoding gene BphS;

b) synthetic bacteria photosensitive diguanylate cyclase BphS encoding gene is linked to the c-di-GMP degrading enzyme YhjH encoding gene by the 2A sequence BphS-2A-YhjH;

c) synthetic bacteria photosensitive diguanylate cyclase BphS encoding gene is linked to the phytochrome synthase BphO encoding gene by 2A sequence Bphs-2A-BphO;

d) Synthetic bacteria Photosensitive diguanylate cyclase BphS encoding gene is linked to the phytochrome synthase BphO encoding gene by 2A sequence, and then linked to the c-di-GMP degrading enzyme YhjH encoding gene by 2A sequence BphS-2A- BphO-2A-YhjH;

Wherein the 2A sequence can be replaced by an internal ribosome entry site sequence IRES;

The phytochrome synthase BphO has a function of synthesizing a phytochrome biliverdin;

The c-di-GMP degrading enzyme YhjH has a function of degrading c-di-GMP to pGpG;

The amino acid sequences of BphS and BphO are shown in SEQ ID NO. 15, and SEQ ID NO. 16, respectively, and the amino acid sequence of YhjH is GeneBank accession number NP_417982.
The far red light gene loop expression control system according to claim 12, wherein said processor comprises: an immune signal transduction molecule STING; or said processor is a DNA binding domain and c-di- a complex of a GMP binding domain polypeptide, a polypeptide as a nuclear localization signal NLS, a polypeptide as a linker domain, and a polypeptide as a transcriptional regulatory domain;

The immune signaling molecule STING has the nucleotide sequence Genebank accession number: NM_198282; the polypeptide as a DNA binding domain and a c-di-GMP binding domain, which is capable of binding to c-di-GMP a DNA sequence-binding protein, including a BldD protein, the amino acid sequence of which is set forth in SEQ ID NO.

The polypeptide as the nuclear localization signal NLS, which may be in the form of 1-3 copies, and the amino acid sequence thereof is shown in SEQ ID NO.

The polypeptide as a linking domain may have a length from 0-30 amino acids in multiple forms, and the amino acid sequence thereof is shown in SEQ ID NO.

The polypeptide as a transcriptional regulatory domain, which is a domain protein having a transcriptional activation function;

The polypeptide as a transcriptional regulatory domain is placed at the N-terminus or C-terminus of the polypeptide BldD of the DNA-binding domain and the c-di-GMP binding domain.
The far red light gene loop expression control system according to claim 12, wherein the effector comprises a promoter P FRL and a gene gene reporter, denoted as P FRL -reporter; wherein the promoter P FRL Including a DNA sequence recognized and bound by IFR3 and a weak promoter, the nucleotide sequence is selected from any one of SEQ ID NOs. 6 to 14; or, the promoter P FRL includes a BldD protein-binding DNA sequence and a promoter gene. a weak promoter expressed, the nucleotide sequence being selected from any one of SEQ ID NOs. 23-42; wherein the processor BldD protein binds to a DNA sequence which is a DNA binding domain and c-di-GMP binding The DNA sequence specifically recognized and bound by the polypeptide of the domain is a partial sequence of the bldM promoter region, the nucleotide sequence thereof is SEQ ID NO. 21, and is a partial sequence of the whiG promoter region, and the nucleotide sequence is as SEQ. ID NO.22, and different combinations of different copy numbers, bldM and whiG of bldM and whiG;

The weak promoter for promoter gene expression includes all weak promoters including TATAbox, the cytomegalovirus hCMV minimal promoter and its mutant hCMVmin 3G.
Eukaryotic expression containing the far red light gene loop expression control system according to any one of claims 12-16 Expression vector, engineered cell or engineered cell transplantation vector.
Use of a far red light gene loop expression control system or a eukaryotic expression vector containing the far red light gene loop expression control system for preparing a diabetes therapeutic drug/product, characterized in that the diabetes includes type I Diabetes and / or type 2 diabetes.
The use according to claim 18, wherein said far red light gene loop expression control system regulates expression of insulin and/or glucagon-like peptide GLP-1; said insulin expression construct comprises SEAP- 2A-Insulin, EGFP-2A-Insulin, EGFP-2A-SEAP-2A-Insulin; expression of the glucagon-like peptide GLP-1 includes GLP-1-Fc.