CN114324860A - A method for assessing the correlation between plasmablasts and pancreatic islet immune damage - Google Patents

Abstract

The invention discloses a method for assessing the correlation between plasmablasts and pancreatic islet immune damage, including separation of tissue samples, flow cytometry analysis and sorting of plasmablasts, cell co-culture and/or cell adoptive transfer, and establishment of an assessment standard for insulitis Wait. The evaluation method of the present invention can effectively establish the correlation between plasmablasts and pancreatic islet immune damage, and provides a possibility for early diagnosis of immune damage in type 1 diabetes.

Description

A method for assessing the correlation between plasmablasts and pancreatic islet immune damage

technical field

The invention belongs to the technical field of biology and medicine, and particularly relates to a method for evaluating the correlation between plasmablasts and pancreatic islet immune damage.

Background technique

Type 1 diabetes is an autoimmune disease characterized by absolute insulin deficiency caused by pancreatic β-cell-specific immune damage. Once the patient is diagnosed, exogenous insulin therapy should be started as soon as possible to reduce the metabolic load of pancreatic beta cells and reduce the risk of diabetic ketosis or ketoacidosis. A clear diagnosis of diabetes type and early identification of type 1 diabetes are crucial for formulating treatment plans. At present, there is still no clear diagnostic criteria for type 1 diabetes, which is mainly based on clinical manifestations. There are few laboratory indicators for auxiliary diagnosis, mainly referring to the determination of islet autoantibodies, serum insulin or C-peptide. However, the clinical characteristics of the type 1 diabetes population are heterogeneous, and there are large differences in the age of onset, onset characteristics, islet functional status, and the type of islet autoantibodies. The diagnosis of some patients is often a retrospective diagnosis. Diagnosis in the early stages of the disease is sometimes very difficult.

The only detection indicators to assist clinical diagnosis are islet autoantibodies and islet function assays. It is urgent to find new methods that can effectively reflect islet autoimmune damage. At present, a B lymphocyte subtype involved in islet immune damage in type 1 diabetes has been found, but the correlation between plasmablasts and islet immune damage is not known, and it is impossible to assess the islet immune damage caused by plasmablasts in type 1 diabetes. effect.

SUMMARY OF THE INVENTION

In view of this, the present invention expects to provide a method for assessing the correlation between plasmablasts and pancreatic islet immune damage, which can help to explore the correlation between plasmablasts and pancreatic islet immune damage, and the effect of plasmablasts on islet immune damage in type 1 diabetes. effect for an accurate assessment.

In order to achieve the above object, the technical scheme of the present invention is achieved in this way:

The invention provides a method for evaluating the correlation between plasmablasts and pancreatic islet immune damage, comprising the following steps:

1) Isolate the immune cells in the pancreatic lymph node tissue and spleen of different stages of type 1 diabetes patients or NOD model animals; preferably, the NOD model animals of different stages are raised to 4 weeks, 8 weeks, 12 weeks, 16 weeks, 20 weeks. NOD model animals for 24 weeks; preferably, the NOD model animals are NOD mice;

2) Immune cell analysis, sorting plasmablasts, and constructing the relationship between the content of plasmablasts and different stages of type 1 diabetes patients or NOD model animals is positive correlation;

3) Adopt a combination of plasmablasts or plasmablasts and T lymphocytes to carry out adoptive transfer, monitor the rate and proportion of diabetes, and the situation of insulitis;

And/or, co-culture T lymphocytes, plasmablasts and islets to detect islet apoptosis;

4) To establish the evaluation criteria for insulitis, and to score the insulitis according to the degree of immune cell infiltration in the islets.

The invention also provides insulitis scoring criteria: 0 points, complete islets, no immune cell infiltration; 1 point, immune cell infiltration only around the islets, or the immune cell infiltration area in the islets is less than 25%; 2 points, immune cells in the islets The infiltrated area is 25%-50%; 3 points, the immune cell infiltration area in the islet is 50%-75%; 4 points, the immune cell infiltration area in the islet exceeds 75%.

The step 1) includes separating pancreatic lymph nodes and spleen, grinding to obtain a cell suspension, and centrifuging the pancreatic lymph node cell suspension to obtain pancreatic lymph node immune cells; adding red blood cell lysate to the spleen cell precipitation, and centrifuging to obtain spleen immune cells after lysis.

The step 2) includes transferring the immune cells of step 1) to a flow tube, adding a flow antibody premix to incubate, then adding a cell death dye to incubate, washing and resuspending in Stain buffer, and sorting using a flow cytometer. required T lymphocytes and plasmablasts.

The step 3) includes co-transferring T lymphocytes and plasmablasts to NOD/SCID mice, monitoring random blood glucose, obtaining pancreatic tissue, sectioning, performing HE staining and immunohistochemical staining, and collecting pictures.

In the step 3), the ratio of T lymphocytes to plasmablasts is 1-10:10-1, preferably 20:2-5 or 2:1.

The method for detecting islet apoptosis in the step 3) is the TUNEL method.

The co-transfer of T lymphocytes and plasmablasts is carried out by a uniform bolus injection from the fundus venous plexus; the random blood glucose monitoring is monitoring the random blood glucose of NOD/SCID mice by the tail clipping method.

The present invention also provides the use of plasmablasts for preparing islet immune-injuring agent, the islet immune-injuring agent optionally further comprising T lymphocytes, wherein the ratio of T lymphocytes to plasmablasts is 1-1 10:10-1, preferably 2:1 or 20:2-5.

Specifically, the present invention also provides a method for evaluating the correlation between plasmablasts and pancreatic islet immune damage, comprising the following steps:

Isolation of tissue samples: The pancreatic lymph nodes, pancreas, and spleen of NOD mice were isolated.

Immune cell preparation and islet culture: Grind the spleen and pancreatic lymph nodes of NOD mice to obtain a cell suspension, add cell lysate for lysis, and centrifuge to obtain immune cells for later use. Collagenase was added to digest the pancreas, and then the digestion was terminated with serum-containing HBSS solution. The islets without fragments were selected under a stereomicroscope and cultured in the culture medium.

Flow cytometry analysis: Transfer immune cells to flow tubes, add flow antibody premix and incubate in the dark, then add cell dead dye and incubate in the dark. After washing and resuspending in Stain buffer, use flow cytometer for analysis. analyze.

Preparation of T lymphocytes and plasmablasts: Take immune cells and use flow cytometry to aseptically sort T lymphocytes and plasmablasts.

Cell co-culture: Use a 96-well round-bottom cell culture plate to culture in 3 groups with 20-30 islets/well, and add 2×10 ⁵ T lymphocytes and 1×10 ⁵ plasmablasts to the first group. Pancreatic islets were co-cultured. Group 2 was added with 2×10 ⁵ T lymphocytes to co-culture with islets. Group 3 was cultured with islets alone. 10ng/mL of IL-2 was added to each well. After cell culture, TUNEL assay was used to detect islet apoptosis. And/or, adoptive transfer of cells: NOD/SCID mice were randomly divided into 3 groups, group 1 received NOD mice 2 x 10 ⁶ T lymphocytes and 2 x 10 ⁵ -5 x 10 ⁵ plasmablasts each Co-transfer, each of the second group received the same number of T lymphocytes transplantation, and the third group received an equal volume of PBS injection; after cell transplantation, the random blood glucose of the three groups of NOD/SCID mice was monitored; NOD/SCID mice were sacrificed after the onset of the disease. Pancreatic HE staining, microscopic examination, and insulitis score were performed according to the degree of immune cell infiltration in the islets.

Here, the extraction of immune cells and the culture of pancreatic islets were performed on NOD mice at different culture periods, which was beneficial for gradient control and convenient for analysis.

Further, the specific operation method of the cell co-cultivation is to gently shake the culture plate by drawing a circle, so that the islets and the immune cells are aggregated in the middle, directly contacted, and placed in a 37°C, 5% CO2 incubator for 72 hours. Aspirate the mixture in each well and wash it in an EP tube containing 1 mL of PBS. The islets will settle at the bottom of the EP tube. The supernatant was carefully aspirated and discarded, washed again, and the apoptosis of islets was detected by TUNEL method.

Furthermore, T lymphocytes and plasmablasts were extracted from 16-week-old NOD mice in adoptive cell transfer, and the cells were transplanted by a uniform bolus injection from the fundus venous plexus; the random blood glucose of NOD/SCID mice was monitored by tail clipping method.

Further, the insulitis scoring criteria are: 0 points, complete islets, no immune cell infiltration; 1 point, immune cell infiltration only around the islets, or the immune cell infiltration area in the islets is less than 25%; 2 points, in the islets The area of immune cell infiltration reaches 25%-50%; 3 points, the immune cell infiltration area in the pancreatic islet reaches 50%-75%; islets were scored.

In the present invention, the insulitis scoring standard can assist in exploring the correlation between plasmablasts and islet immune damage, and accurately evaluate the role of plasmablasts in islet immune damage in type 1 diabetes.

The beneficial effects of the present invention are as follows: 1) The method for evaluating the correlation between plasmablasts and pancreatic islet immune damage of the present invention can assist in exploring the correlation between plasmablasts and pancreatic islet immune damage, and can assist in the investigation of the correlation between plasmablasts and islet immune damage in type 1 diabetes mellitus. 2) The method of the present invention co-cultures plasmablasts and T lymphocytes with pancreatic islets, and adopts adoptive transfer of plasmablasts and T lymphocytes, which fully shows that plasmablasts and T lymphocytes respond synergistically, The effect of leading to islet immune damage provides the possibility for the early diagnosis of immune damage in type 1 diabetes; 3) Plasmablasts can be used as islet immune damage agents.

Description of drawings

Fig. 1 is the proportion of plasmablasts in the spleen of mice according to the embodiment of the present invention;

Fig. 2 is the proportion of plasmablasts in the pancreatic lymph node of the mouse according to the embodiment of the present invention;

Fig. 3 is the infiltration of plasmablasts in mouse islets of the embodiment of the present invention;

Fig. 4 is the embodiment of the present invention TUNEL method detects islet apoptosis under different culture conditions;

Fig. 5 is the blood sugar changes of 16-week-old NOD/SCID mice according to the embodiment of the present invention;

Figure 6 shows the degree of insulitis in 16-week-old NOD/SCID mice according to the embodiment of the present invention.

Detailed ways

In order to be able to understand the features and technical content of the present invention in more detail, the implementation of the present invention is described in detail below with reference to the accompanying drawings, which are for reference only and are not intended to limit the present invention.

Example 1

Immune cell extraction:

1) Isolation of pancreatic lymph nodes, pancreas, and spleen of NOD mice: NOD mice were fed to 4 weeks, 8 weeks, 12 weeks, 16 weeks, 20 weeks, and 24 weeks, and then sacrificed in batches. The mice were fixed on a surgical board, and the abdomen was wetted with an alcohol swab. Cut the fur and muscle layer layer by layer to expose the internal organs. A pair of oval lymph nodes along the blood vessels on the pancreas was carefully isolated, placed in PBS, and placed on ice. Pancreatic tissue was dissociated from the bowel margins and immersed in 4% paraformaldehyde fixative. The spleen was separated from the pancreas, and the spleen was placed in PBS on ice.

2) Extraction of immune cells from mouse spleen and pancreatic lymph nodes: place a disposable sterile sieve in the mouth of a 50 mL centrifuge tube, place the spleen on the sieve, and carefully grind the spleen. Centrifuge the cell suspension in the centrifuge tube and discard the supernatant. Add 1× red blood cell lysate and lyse at room temperature for 10 min. After stopping the lysis, centrifuge and discard the supernatant. Wash the cells once, centrifuge and discard the supernatant to obtain the spleen immune cells at the bottom of the tube.

The extraction of immune cells from pancreatic lymph nodes is the same as that of spleen.

Example 2

To culture islets:

5-week-old mice were anesthetized and fixed, the fur and muscle layer were cut, and the heart was exposed by thoracotomy, taking care to avoid intraperitoneal hemorrhage. Locate the opening of the common pancreatic duct duodenum and ligate it with a thread. The right atrial appendage was cut, the venous blood was drained, the common bile duct was found near the hepatic hilum, the cannula was intubated, and collagenase was injected. The pancreas were carefully dissociated and immediately placed on ice. Add about 5 mL of collagenase V to a 50 mL plastic centrifuge tube containing pancreatic tissue on ice, and digest at 37°C for 26-28 min. Immediately after the digestion, place a 50 mL plastic centrifuge tube on ice, vortex until the tissue becomes sandy, 5 seconds/time, 3 times in total; add 2 times the volume of pre-cooled serum-containing HBSS solution to terminate the digestion. Pass through a 30-mesh sieve, remove the undigested tissue pieces, wash with HBSS three times, discard the supernatant, add 5 mL of Histopaque, mix by pipetting, slowly add 5 mL of HBSS along the tube wall, and the solution is separated into two layers. After centrifugation, carefully aspirate the white sandwich layer in the middle of the centrifuge tube with a 1mL pipette and transfer it to a petri dish containing serum HBSS. Select the intact, moderately sized, and fragment-free islets under a stereomicroscope and place them in an RPMI 1640+ 10% FBS + double antibody in the culture medium.

Example 3

Immune cell analysis:

The spleen and lymph node immune cells of Example 1 were transferred to a flow tube. The mouse anti-B220 (PerCP-Cy5.5), anti-CD138 (APC), anti-CD44 (BB515), and anti-CD3 (PE-Cy7) antibodies were pre-mixed and added to the flow tube. Cells were mixed and incubated at room temperature for 14-20 minutes in the dark. Add 1 mL of PBS, centrifuge at 400 g for 5 min, and discard the supernatant. Then add 1 mL of PBS and 1 μl of Fixable viability stain (FVS) 780 cell death dye, and incubate at room temperature for 10-15 min in the dark. Add 1 mL of Stain buffer (PBS+1% FBS), centrifuge at 400 g for 5 min, and discard the supernatant. The cells obtained at the bottom of the tube were resuspended with 300 μl of Stain buffer (PBS+1% FBS). Analysis was performed using a BD FACSAira III flow cytometer and the resulting data were analyzed using FlowJo 10.4 software.

Figure 1 shows the proportion of plasmablasts in the spleen of mice according to the embodiment of the present invention. As shown in Figure 1, before the occurrence of insulitis, the proportion of plasmablasts in the spleen of NOD mice was low. The proportion of plasmablasts in the spleen of 16w NOD mice was significantly higher than that of 4w NOD mice.

Figure 2 shows the proportion of plasmablasts in the pancreatic lymph nodes of the mice according to the embodiment of the present invention. As shown in Figure 2, before the occurrence of insulitis, the proportion of plasmablasts in the pancreatic lymph nodes of NOD mice was low, and as the insulitis progressed, At 8w, 12w and 16w, the proportion of plasmablasts increased significantly.

Figure 3 shows the infiltration of plasmablasts in the pancreatic islets of the mice according to the embodiment of the present invention. As shown in Figure 3, plasmablasts infiltration appeared in the islets of 4w and 16w NOD mice, and the degree of insulitis in 16w NOD mice was aggravated. Cell infiltration was significantly increased.

The above results show that plasmablasts are involved in islet autoimmunity damage and type 1 diabetes in NOD mice, and plasmablasts are positively correlated with the degree of islet immune damage.

Example 4

To prepare T lymphocytes and plasmablasts:

Immune cells extracted from the spleen and pancreatic lymph nodes of 5-week-old and 16-week-old NOD mice were collected according to the method in Example 1, and T lymphocytes and plasmablasts were aseptically sorted using a BD FACSAira III flow cytometer.

Example 5

Cell co-culture:

Use a 96-well round-bottom cell culture plate to culture in 3 groups with 20-30 islets/well. The first group is the experimental group, which adds 2 × 10 ⁵ T lymphocytes and 1 × 10 ⁵ plasmablasts , co-cultured with islets; in group 2, 2×10 ⁵ T lymphocytes were added to co-culture with islets; in group 3, islets were cultured alone. Groups 2 and 3 were control groups. 10ng/mL of IL-2 was added to each well. Gently shake the culture plate in a circular motion to make the islets and immune cells gather in the middle and make direct contact. After culturing in a 37°C, 5% CO ₂ incubator for 72 hours, aspirate the mixture in the culture wells and place them in 1 mL PBS. After washing in the EP tube, the islets will settle to the bottom of the EP tube. The supernatant was carefully aspirated and discarded, washed again, and the apoptosis of islets was detected by TUNEL method.

Figure 4 is an example of the TUNEL method for detecting islet apoptosis under different culture conditions. As shown in Figure 4, a co-culture system of mouse pancreatic islets and plasmablasts or/and T lymphocytes was established in vitro, using TUNEL reagent It was found that compared with the culture conditions without plasmablasts, the apoptosis of islets was significantly increased when the islets were co-cultured with plasmablasts and T lymphocytes, indicating that plasmablasts and T lymphocytes responded synergistically, resulting in a significant increase in islet apoptosis. Islet immune damage.

Example 6

Adoptive transfer of cells:

T lymphocytes and plasmablasts of 16-week-old NOD mice were collected, and the cells were transplanted according to the following cell amounts. 6-week-old NOD/SCID mice were randomly divided into 3 groups with 6 mice in each group. The first group was the experimental group, each received 2×10 ⁶ T lymphocytes and 2×10 ⁵ -5×10 ⁵ plasmablasts from 16-week-old NOD mice; the second group received 20-week-old mice each NOD mice were transplanted with the same number of T lymphocytes, and the third group was injected with equal volume of PBS. Groups 2 and 3 were control groups.

Transplantation method: NOD/SCID mice were anesthetized with inhaled isoflurane. Fix the head of the mouse, suck the transplanted cell suspension with an insulin syringe, insert it into the fundus venous plexus along the inner canthus of the mouse slightly, inject at a constant speed, and slowly pull out the syringe after staying for a few seconds. After cell transplantation, the random blood glucose of NOD/SCID mice in three groups was monitored by tail clipping method. NOD/SCID mice were sacrificed after the onset of disease, pancreas HE staining was performed, and microscopic examination was performed.

Insulitis evaluation criteria (total score of each islet/total number of observed islets): 0 points, complete islets, no immune cell infiltration; 1 point, immune cell infiltration only around the islets, or the immune cell infiltration area in the islets is less than 25%; 2 25%-50% of the islets infiltrated; 3 points, 50%-75% of the islets infiltrated; 4 points, more than 75% of the islets infiltrated; NOD/SCID in each group was small. Mice observe at least 100 islets for scoring.

In the cell adoptive transfer experiment, by monitoring blood glucose, the rate and proportion of the three groups of recipient mice developing diabetes were compared. Figure 5 is the blood sugar changes of 16-week-old NOD/SCID mice according to the example of the present invention. As shown in Figure 5, the NOD/SCID mice that received co-transfer of plasmablasts and T lymphocytes started to develop diabetes 32 days after transplantation , while the 2 control NOD/SCID mice that received only T lymphocytes or PBS injections remained normoglycemic 80 days after adoptive transfer.

Figure 6 shows the degree of insulitis in 16-week-old NOD/SCID mice according to the example of the present invention. The insulitis in the mice was evaluated by pancreas HE staining. As shown in Figure 6, the degree of insulitis in the three groups of recipient mice showed obvious Differences. Most of the islets in the mice receiving plasmablast and T lymphocyte co-transfer were infiltrated by a large number of immune cells, and the degree of insulitis was more severe than the other two groups. These results indicate that plasmablasts are pathogenic cells that lead to islet immune damage and type 1 diabetes.

Here, in step 5) cell co-culture experiment and step 6) cell adoptive transfer experiment, the experimental results fully show that plasmablasts and T lymphocytes respond synergistically to cause immune damage to islets, which is the early stage of immune damage in type 1 diabetes. Diagnosis offers the possibility.

The NOD mice (non-obese diabetic mice) mentioned above were SPF grade, 4-20 weeks old, female; NOD/SCID mice (severe combined immunodeficiency NOD mice) were SPF grade, 6 weeks old, female.

The method of the invention shows through animal model studies that plasmablasts can be used to evaluate islet immune damage in type 1 diabetes, provide an evaluation index for islet immune damage and its degree, and also provide clinical use for plasmablasts to prepare and detect islet immunity. Damaged agents and drugs for the treatment of islet immune damage provide a rationale.

The above are only preferred embodiments of the present invention, and are not intended to limit the protection scope of the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention shall be included in the within the protection scope of the present invention.

Claims (10)

1. a method for assessing the correlation between plasmablasts and pancreatic islet immune damage, is characterized in that, comprises the following steps: 1) Isolate the immune cells in the pancreatic lymph node tissue and spleen of different stages of type 1 diabetes patients or NOD model animals; preferably, the NOD model animals of different stages are raised to 4 weeks, 8 weeks, 12 weeks, 16 weeks, 20 weeks. NOD model animals for 24 weeks; preferably, the NOD model animals are NOD mice; 2) Immune cell analysis, sorting plasmablasts, and constructing the relationship between the content of plasmablasts and different stages of type 1 diabetes patients or NOD model animals is positive correlation; 3) Adopt a combination of plasmablasts or plasmablasts and T lymphocytes to carry out adoptive transfer, monitor the rate and proportion of diabetes, and the situation of insulitis; And/or, co-culture T lymphocytes, plasmablasts and islets to detect islet apoptosis; 4) To establish the evaluation criteria for insulitis, and to score the insulitis according to the degree of immune cell infiltration in the islets. 2 . The method according to claim 1 , wherein the scoring criteria for insulitis: 0 points, complete islets, no immune cell infiltration; 1 point, immune cell infiltration only around the islets, or immune cell infiltration area in the islets Less than 25%; 2 points, the area of immune cell infiltration in the islet reaches 25%-50%; 3 points, the area of immune cell infiltration in the islet reaches 50%-75%; 4 points, the area of immune cell infiltration in the islet exceeds 75%. 3. method according to claim 1, is characterized in that, described step 1) comprises separating pancreatic lymph node and spleen, grinding to obtain cell suspension, after pancreatic lymph node cell suspension is centrifuged to obtain pancreatic lymph node immune cells; spleen cell precipitation The erythrocyte lysate was added to the lysate, and the spleen immune cells were obtained by centrifugation after lysis. 4. The method according to claim 1, wherein the step 2) comprises transferring the immune cells of the step 1) to a flow tube, adding a flow antibody premix to incubate, and then adding a cell death dye to incubate, After washing and resuspending in Stain buffer, the desired T lymphocytes and plasmablasts were sorted by flow cytometry. 5. method according to claim 1, is characterized in that, in described step 3), comprise T lymphocyte and plasmablast are jointly transferred to NOD/SCID mouse, monitor random blood sugar, obtain pancreatic tissue, slice, carry out. HE staining and immunohistochemical staining were used to collect pictures. The method according to claim 1, wherein the ratio of T lymphocytes to plasmablasts in the step 3) is 1-10:10-1, preferably 20:2-5 or 2:1 . 7 . The method according to claim 1 , wherein the method for detecting pancreatic islet apoptosis in the step 3) is the TUNEL method. 8 . 8. The method according to claim 5, wherein the co-transfer of T lymphocytes and plasmablasts is a uniform bolus injection from the fundus venous plexus; the monitoring of random blood glucose is to monitor NOD/SCID by tail clipping method Random blood glucose in mice. 9. Use of a plasmablast for preparing an islet immune-injuring agent, optionally, the islet immune-injuring agent may further comprise T lymphocytes. 10. The use of plasmablasts as claimed in claim 9 for preparing islet immune-injuring agents, wherein the ratio of the T lymphocytes to the plasmablasts is 1 to 10:10 to 1, preferably 2: 1 or 20: 2 to 5.

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